The contribution of airway smooth muscle to airway narrowing and airway hyperresponsiveness in disease.

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Martin, J G; Duguet, A; Eidelman, D H

2000-08-01

Airway hyperresponsiveness (AHR), the exaggerated response to constrictor agonists in asthmatic subjects, is incompletely understood. Changes in either the quantity or properties of airway smooth muscle (ASM) are possible explanations for AHR. Morphometric analyses demonstrate structural changes in asthmatic airways, including subepithelial fibrosis, gland hyperplasia/hypertrophy, neovascularization and an increase in ASM mass. Mathematical modelling of airway narrowing suggests that, of all the changes in structure, the increase in ASM mass is the most probable cause of AHR. An increase in ASM mass in the large airways is more closely associated with a greater likelihood of dying from asthma than increases in ASM mass in other locations within the airway tree. ASM contraction is opposed by the elastic recoil of the lungs and airways, which appears to limit the degree of bronchoconstriction in vivo. The cyclical nature of tidal breathing applies stresses to the airway wall that enhance the bronchodilating influence of the lung tissues on the contracting ASM, in all probability by disrupting cross-bridges. However, the increase in ASM mass in asthma may overcome the limitation resulting from the impedances to ASM shortening imposed by the lung parenchyma and airway wall tissues. Additionally, ASM with the capacity to shorten rapidly may achieve shorter lengths and cause a greater degree of bronchoconstriction when stimulated to contract than slower ASM. Changes in ASM properties are induced by the process of sensitization and allergen-exposure such as enhancement of phospholipase C activity and inositol phosphate turnover, and increases in myosin light chain kinase activity. Whether changes in ASM mass or biochemical/biomechanical properties form the basis for asthma remains to be determined.

Allergic sensitization enhances the contribution of Rho-kinase to airway smooth muscle contraction

NARCIS (Netherlands)

Schaafsma, D.; Gosens, Reinout; Bos, I.S.T.; Meurs, Herman; Zaagsma, Hans; Nelemans, Herman

2004-01-01

1 Repeated allergen challenge has been shown to increase the role of Rho-kinase in airway smooth muscle (ASM) contraction. We considered the possibility that active allergic sensitization by itself, that is, without subsequent allergen exposure, could be sufficient to enhance Rho-kinase-mediated ASM

Arginase strongly impairs neuronal nitric oxide-mediated airway smooth muscle relaxation in allergic asthma

NARCIS (Netherlands)

Maarsingh, H; Leusink, J; Bos, I Sophie T; Zaagsma, J; Meurs, H

2006-01-01

Background: Using guinea pig tracheal preparations, we have recently shown that endogenous arginase activity attenuates inhibitory nonadrenergic noncholinergic (iNANC) nerve-mediated airway smooth muscle relaxation by reducing nitric oxide (NO) production - due to competition with neuronal

The actin regulator zyxin reinforces airway smooth muscle and accumulates in airways of fatal asthmatics.

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Sonia R Rosner

Full Text Available Bronchospasm induced in non-asthmatic human subjects can be easily reversed by a deep inspiration (DI whereas bronchospasm that occurs spontaneously in asthmatic subjects cannot. This physiological effect of a DI has been attributed to the manner in which a DI causes airway smooth muscle (ASM cells to stretch, but underlying molecular mechanisms-and their failure in asthma-remain obscure. Using cells and tissues from wild type and zyxin-/- mice we report responses to a transient stretch of physiologic magnitude and duration. At the level of the cytoskeleton, zyxin facilitated repair at sites of stress fiber fragmentation. At the level of the isolated ASM cell, zyxin facilitated recovery of contractile force. Finally, at the level of the small airway embedded with a precision cut lung slice, zyxin slowed airway dilation. Thus, at each level zyxin stabilized ASM structure and contractile properties at current muscle length. Furthermore, when we examined tissue samples from humans who died as the result of an asthma attack, we found increased accumulation of zyxin compared with non-asthmatics and asthmatics who died of other causes. Together, these data suggest a biophysical role for zyxin in fatal asthma.

Arginase strongly impairs neuronal nitric oxide-mediated airway smooth muscle relaxation in allergic asthma

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Zaagsma Johan

2006-01-01

Full Text Available Abstract Background Using guinea pig tracheal preparations, we have recently shown that endogenous arginase activity attenuates inhibitory nonadrenergic noncholinergic (iNANC nerve-mediated airway smooth muscle relaxation by reducing nitric oxide (NO production – due to competition with neuronal NO-synthase (nNOS for the common substrate, L-arginine. Furthermore, in a guinea pig model of allergic asthma, airway arginase activity is markedly increased after the early asthmatic reaction (EAR, leading to deficiency of agonist-induced, epithelium-derived NO and subsequent airway hyperreactivity. In this study, we investigated whether increased arginase activity after the EAR affects iNANC nerve-derived NO production and airway smooth muscle relaxation. Methods Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz-induced relaxation was measured in tracheal open-ring preparations precontracted to 30% with histamine in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to EFS-induced relaxation was assessed by the nonselective NOS inhibitor Nω-nitro-L-arginine (L-NNA, 100 μM, while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA, 10 μM. Furthermore, the role of substrate availability to nNOS was measured in the presence of exogenous L-arginine (5.0 mM. Results At 6 h after ovalbumin-challenge (after the EAR, EFS-induced relaxation (ranging from 3.2 ± 1.1% at 0.5 Hz to 58.5 ± 2.2% at 16 Hz was significantly decreased compared to unchallenged controls (7.1 ± 0.8% to 75.8 ± 0.7%; P P P Conclusion The results clearly demonstrate that increased arginase activity after the allergen-induced EAR contributes to a deficiency of iNANC nerve-derived NO and decreased airway smooth muscle relaxation, presumably via increased substrate competition with nNOS.

The extracellular matrix deposited by asthmatic airway smooth muscle cells in a resting state reflects a healthy matrix

NARCIS (Netherlands)

Harkness, Louise; Ashton, Anthony; Burgess, Janette

2015-01-01

Introduction: The remodelled asthmatic airway features an altered extracellular matrix (ECM) & increased vasculature. Previous studies found asthmatic (A) airway smooth muscle cells (ASMCs) to deposit an ECM with enhanced bioactivity. These studies however investigated ECM deposited in the presence

Increased proinflammatory responses from asthmatic human airway smooth muscle cells in response to rhinovirus infection

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King Nicholas JC

2006-05-01

Full Text Available Abstract Background Exacerbations of asthma are associated with viral respiratory tract infections, of which rhinoviruses (RV are the predominant virus type. Airway smooth muscle is important in asthma pathogenesis, however little is known about the potential interaction of RV and human airway smooth muscle cells (HASM. We hypothesised that rhinovirus induction of inflammatory cytokine release from airway smooth muscle is augmented and differentially regulated in asthmatic compared to normal HASM cells. Methods HASM cells, isolated from either asthmatic or non-asthmatic subjects, were infected with rhinovirus. Cytokine production was assayed by ELISA, ICAM-1 cell surface expression was assessed by FACS, and the transcription regulation of IL-6 was measured by luciferase activity. Results RV-induced IL-6 release was significantly greater in HASM cells derived from asthmatic subjects compared to non-asthmatic subjects. This response was RV specific, as 5% serum- induced IL-6 release was not different in the two cell types. Whilst serum stimulated IL-8 production in cells from both subject groups, RV induced IL-8 production in only asthmatic derived HASM cells. The transcriptional induction of IL-6 was differentially regulated via C/EBP in the asthmatic and NF-κB + AP-1 in the non-asthmatic HASM cells. Conclusion This study demonstrates augmentation and differential transcriptional regulation of RV specific innate immune response in HASM cells derived from asthmatic and non-asthmatics, and may give valuable insight into the mechanisms of RV-induced asthma exacerbations.

S1P-induced airway smooth muscle hyperresponsiveness and lung inflammation in vivo: molecular and cellular mechanisms.

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Roviezzo, F; Sorrentino, R; Bertolino, A; De Gruttola, L; Terlizzi, M; Pinto, A; Napolitano, M; Castello, G; D'Agostino, B; Ianaro, A; Sorrentino, R; Cirino, G

2015-04-01

Sphingosine-1-phosphate (S1P) has been shown to be involved in the asthmatic disease as well in preclinical mouse experimental models of this disease. The aim of this study was to understand the mechanism(s) underlying S1P effects on the lung. BALB/c, mast cell-deficient and Nude mice were injected with S1P (s.c.) on days 0 and 7. Functional, molecular and cellular studies were performed. S1P administration to BALB/c mice increased airway smooth muscle reactivity, mucus production, PGD2 , IgE, IL-4 and IL-13 release. These features were associated to a higher recruitment of mast cells to the lung. Mast cell-deficient Kit (W) (-sh/) (W) (-sh) mice injected with S1P did not display airway smooth muscle hyper-reactivity. However, lung inflammation and IgE production were still present. Treatment in vivo with the anti-CD23 antibody B3B4, which blocks IgE production, inhibited both S1P-induced airway smooth muscle reactivity in vitro and lung inflammation. S1P administration to Nude mice did not elicit airway smooth muscle hyper-reactivity and lung inflammation. Naïve (untreated) mice subjected to the adoptive transfer of CD4+ T-cells harvested from S1P-treated mice presented all the features elicited by S1P in the lung. S1P triggers a cascade of events that sequentially involves T-cells, IgE and mast cells reproducing several asthma-like features. This model may represent a useful tool for defining the role of S1P in the mechanism of action of currently-used drugs as well as in the development of new therapeutic approaches for asthma-like diseases. © 2014 The British Pharmacological Society.

A 'Good' muscle in a 'Bad' environment: the importance of airway smooth muscle force adaptation to airway hyperresponsiveness.

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Bossé, Ynuk; Chapman, David G; Paré, Peter D; King, Gregory G; Salome, Cheryl M

2011-12-15

Asthma is characterized by airway inflammation, with a consequent increase in spasmogens, and exaggerated airway narrowing in response to stimuli, termed airway hyperresponsiveness (AHR). The nature of any relationship between inflammation and AHR is less clear. Recent ex vivo data has suggested a novel mechanism by which inflammation may lead to AHR, in which increased basal ASM-tone, due to the presence of spasmogens in the airways, may "strengthen" the ASM and ultimately lead to exaggerated airway narrowing. This phenomenon was termed "force adaptation" [Bossé, Y., Chin, L.Y., Paré, P.D., Seow, C.Y., 2009. Adaptation of airway smooth muscle to basal tone: relevance to airway hyperresponsiveness. Am. J. Respir. Cell Mol. Biol. 40, 13-18]. However, it is unknown whether the magnitude of the effect of force adaptation ex vivo could contribute to exaggerated airway narrowing in vivo. Our aim was to utilize a computational model of ASM shortening in order to quantify the potential effect of force adaptation on airway narrowing when all other mechanical factors were kept constant. The shortening in the model is dictated by a balance between physiological loads and ASM force-generating capacity at different lengths. The results suggest that the magnitude of the effect of force adaptation on ASM shortening would lead to substantially more airway narrowing during bronchial challenge at any given airway generation. We speculate that the increased basal ASM-tone in asthma, due to the presence of inflammation-derived spasmogens, produces an increase in the force-generating capacity of ASM, predisposing to AHR during subsequent challenge. Copyright © 2011 Elsevier B.V. All rights reserved.

Ca2+ sparks act as potent regulators of excitation-contraction coupling in airway smooth muscle.

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Zhuge, Ronghua; Bao, Rongfeng; Fogarty, Kevin E; Lifshitz, Lawrence M

2010-01-15

Ca2+ sparks are short lived and localized Ca2+ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca2+-activated K+ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca2+ channels. But in many smooth muscles from a variety of organs, Ca2+ sparks can additionally activate Ca2+-activated Cl(-) channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca2+ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca2+ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca2+ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca2+ channels, resulting in an increase in global [Ca2+](i) and cell contraction. Therefore, Ca2+ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.

Nogo-B regulates migration and contraction of airway smooth muscle cells by decreasing ARPC 2/3 and increasing MYL-9 expression

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Cai Zailong

2011-01-01

Full Text Available Abstract Background Abnormal proliferation, apoptosis, migration and contraction of airway smooth muscle (ASM cells in airway remodeling in asthma are basically excessive repair responses to a network of inflammatory mediators such as PDGF, but the mechanisms of such responses remain unclear. Nogo-B, a member of the reticulum family 4(RTN4, is known to play a key role in arteriogenesis and tissue repair. Further studies are needed to elucidate the role of Nogo-B in airway smooth muscle abnormalities. Methods A mouse model of chronic asthma was established by repeated OVA inhalation and subjected to Nogo-B expression analysis using immunohistochemistry and Western Blotting. Then, primary human bronchial smooth muscle cells (HBSMCs were cultured in vitro and a siRNA interference was performed to knockdown the expression of Nogo-B in the cells. The effects of Nogo-B inhibition on PDGF-induced HBSMCs proliferation, migration and contraction were evaluated. Finally, a proteomic analysis was conducted to unveil the underlying mechanisms responsible for the function of Nogo-B. Results Total Nogo-B expression was approximately 3.08-fold lower in chronic asthmatic mice compared to naïve mice, which was obvious in the smooth muscle layer of the airways. Interference of Nogo-B expression by siRNA resulted nearly 96% reduction in mRNA in cultured HBSMCs. In addition, knockdown of Nogo-B using specific siRNA significantly decreased PDGF-induced migration of HBSMCs by 2.3-fold, and increased the cellular contraction by 16% compared to negative controls, but had limited effects on PDGF-induced proliferation. Furthermore, using proteomic analysis, we demonstrate that the expression of actin related protein 2/3 complex subunit 5 (ARPC 2/3 decreased and, myosin regulatory light chain 9 isoform a (MYL-9 increased after Nogo-B knockdown. Conclusions These data define a novel role for Nogo-B in airway remodeling in chronic asthma. Endogenous Nogo-B, which may exert

β2-Agonist induced cAMP is decreased in asthmatic airway smooth muscle due to increased PDE4D

NARCIS (Netherlands)

Trian, Thomas; Burgess, Janette K; Niimi, Kyoko; Moir, Lyn M; Ge, Qi; Berger, Patrick; Liggett, Stephen B; Black, Judith L; Oliver, Brian G

2011-01-01

BACKGROUND AND OBJECTIVE: Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known. OBJECTIVE:

Phenotype and Functional Features of Human Telomerase Reverse Transcriptase Immortalized Human Airway Smooth Muscle Cells from Asthmatic and Non-Asthmatic Donors

NARCIS (Netherlands)

Burgess, J. K.; Ketheson, A.; Faiz, A.; Rempel, K. A. Limbert; Oliver, B. G.; Ward, J. P. T.; Halayko, A. J.

2018-01-01

Asthma is an obstructive respiratory disease characterised by chronic inflammation with airway hyperresponsiveness. In asthmatic airways, there is an increase in airway smooth muscle (ASM) cell bulk, which differs from non-asthmatic ASM in characteristics. This study aimed to assess the usefulness

Estimation of airway smooth muscle stiffness changes due to length oscillation using artificial neural network.

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Al-Jumaily, Ahmed; Chen, Leizhi

2012-10-07

This paper presents a novel approach to estimate stiffness changes in airway smooth muscles due to external oscillation. Artificial neural networks are used to model the stiffness changes due to cyclic stretches of the smooth muscles. The nonlinear relationship between stiffness ratios and oscillation frequencies is modeled by a feed-forward neural network (FNN) model. The structure of the FNN is selected through the training and validation using literature data from 11 experiments with different muscle lengths, muscle masses, oscillation frequencies and amplitudes. Data pre-processing methods are used to improve the robustness of the neural network model to match the non-linearity. The validation results show that the FNN model can predict the stiffness ratio changes with a mean square error of 0.0042. Copyright © 2012 Elsevier Ltd. All rights reserved.

Tumor necrosis factor-alpha enhances mRNA expression and secretion of interleukin-6 in cultured human airway smooth muscle cells

NARCIS (Netherlands)

S. McKay (Sue); S.J. Hirst (Stuart); M. Bertrand-de Haas (Marion); J.C. de Jongste (Johan); H.C. Hoogsteden (Henk); P.R. Saxena (Pramod Ranjan); H.S. Sharma (Hari)

2000-01-01

textabstractAirway smooth muscle (ASM) is considered to be an end-target cell for the effects of mediators released during airway wall inflammation. Several reports suggest that activated ASM may be capable of generating various proinflammatory cytokines. We

The laminin beta 1-competing peptide YIGSR induces a hypercontractile, hypoproliferative airway smooth muscle phenotype in an animal model of allergic asthma

NARCIS (Netherlands)

Dekkers, Bart G. J.; Bos, I. Sophie T.; Halayko, Andrew J.; Zaagsma, Johan; Meurs, Herman

2010-01-01

Background: Fibroproliferative airway remodelling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. In vitro studies have shown that maturation of ASM cells to a (hyper)contractile phenotype is dependent on laminin, which can

Membrane Currents in Airway Smooth Muscle: Mechanisms and Therapeutic Implications

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Luke J Janssen

1997-01-01

Full Text Available Electrophysiological and pharmacological techniques were used to characterize the membrane conductance changes underlying spasmogen-evoked depolarization in airway smooth muscle (ASM. Changes included a transient activation of chloride ion channels and prolonged suppression of potassium ion channels; both changes are triggered by release of internally sequestered calcium ion and in turn cause opening of voltage-dependent calcium channels. The resultant influx of calcium ions contributes to contraction as well as to refilling of the internal calcium ion pool. Bronchodilators, on the other hand, act in part through activation of potassium channels, with consequent closure of calcium channels. The tools used to study ion channels in ASM are described, and the investigations of the roles of ion channels in ASM physiology (autacoid-evoked depolarization and hyperpolarization and pathophysiology (airway hyperresponsiveness are summarized. Finally, how the relationship between ion channels and ASM function/dysfunction may relate to the treatment of asthma and related breathing disorders is discussed.

Phosphodiesterases regulate airway smooth muscle function in health and disease.

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Krymskaya, Vera P; Panettieri, Reynold A

2007-01-01

On the basis of structure, regulation, and kinetic properties, phosphodiesterases (PDEs) represent a superfamily of enzymes divided into 11 subfamilies that catalyze cytosolic levels of 3',5'-cyclic adenosine monophosphate (cAMP) or 3',5'-cyclic guanosine monophosphate (cGMP) to 5'-AMP or 5'-GMP, respectively. PDE4 represents the major PDE expressed in inflammatory cells as well as airway smooth muscle (ASM), and selective PDE4 inhibitors provide a broad spectrum of anti-inflammatory effects such as abrogating cytokine and chemokine release from inflammatory cells and inhibiting inflammatory cell trafficking. Due to cell- and tissue-specific gene expression and regulation, PDEs modulate unique organ-based functions. New tools or compounds that selectively inhibit PDE subfamilies and genetically engineered mice deficient in selective isoforms have greatly enhanced our understanding of PDE function in airway inflammation and resident cell function. This chapter will focus on recent advances in our understanding of the role of PDE in regulating ASM function.

A study of airway smooth muscle in asthmatic and non-asthmatic airways using PS-OCT (Conference Presentation)

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Adams, David C.; Holz, Jasmin A.; Szabari, Margit V.; Hariri, Lida P.; Harris, R. Scott; Cho, Jocelyn L.; Hamilos, Daniel L.; Luster, Andrew D.; Medoff, Benjamin D.; Suter, Melissa J.

2016-03-01

Present understanding of the pathophysiological mechanisms of asthma has been severely limited by the lack of an imaging modality capable of assessing airway conditions of asthma patients in vivo. Of particular interest is the role that airway smooth muscle (ASM) plays in the development of asthma and asthma related symptoms. With standard Optical Coherence Tomography (OCT), imaging ASM is often not possible due to poor structural contrast between the muscle and surrounding tissues. A potential solution to this problem is to utilize additional optical contrast factors intrinsic to the tissue, such as birefringence. Due to its highly ordered structure, ASM is strongly birefringent. Previously, we demonstrated that Polarization Sensitive OCT(PS-OCT) has the potential to be used to visualize ASM as well as easily segment it from the surrounding (weakly) birefringent tissue by exploiting a property which allows it to discriminate the orientation of birefringent fibers. We have already validated our technology with a substantial set of histological comparisons made against data obtained ex vivo. In this work we present a comprehensive comparison of ASM distributions in asthmatic and non-asthmatic human volunteers. By isolating the ASM we parameterize its distribution in terms of both thickness and band width, calculated volumetrically over centimeters of airway. Using this data we perform analyses of the asthmatic and non-asthmatic airways using a broad number and variety and subjects.

Relaxation of soman-induced contracture of airway smooth muscle in vitro. (Reannouncement with new availability information)

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Filbert, M.G.; Moore, D.H.; Adler, M.

1992-12-31

A possible role for beta-adrenergic agonists in the management of bronchoconstriction resulting from exposure to anticholinesterase compounds was investigated in vitro in canine tracheal smooth muscle. Norepinephrine, salbutamol and isoproterenol produced partial relaxation of soman-induced contractures. However, the relaxation induced was not sustained; muscle tensions returned to pretreatment levels within minutes despite the continued presence of beta-agonists. Increasing cAMP levels with the non beta-agonist bronchodilators such as thoophylline, a phosphodiesterase inhibitor, or forskolin, a specific stimulator of adenylate cyclase, resulted in more complete and longer lasting relaxation, suggesting that beta-adrenoceptor desensitization may contribute to the failure by beta-agonists to produce sustained relaxation. Nerve agents, Soman, Toxicity, Airway smooth muscle, In vitro, Physiology, Effects.

Bitter Taste Receptors in The Wrong Place: Novel Airway Smooth Muscle Targets For Treating Asthma

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Liggett, Stephen B.

2014-01-01

There is a need to expand the classes of drugs used to treat obstructive lung diseases to achieve better outcomes. With only one class of direct bronchodilators (β-agonists), we sought to find receptors on human airway smooth muscle (ASM) that act via a unique mechanism to relax the muscle, have a diverse agonist binding profile to enhance the probability of finding new therapeutics, and relax ASM with equal or greater efficacy than β-agonists. We have found that human and mouse ASM express s...

Human mast cell and airway smooth muscle cell interactions: implications for asthma.

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Page, S; Ammit, A J; Black, J L; Armour, C L

2001-12-01

Asthma is characterized by inflammation, hyperresponsiveness, and remodeling of the airway. Human mast cells (HMCs) play a central role in all of these changes by releasing mediators that cause exaggerated bronchoconstriction, induce human airway smooth muscle (HASM) cell proliferation, and recruit and activate inflammatory cells. Moreover, the number of HMCs present on asthmatic HASM is increased compared with that on nonasthmatic HASM. HASM cells also have the potential to actively participate in the inflammatory process by synthesizing cytokines and chemokines and expressing surface molecules, which have the capacity to perpetuate the inflammatory mechanisms present in asthma. This review specifically examines how the mediators of HMCs have the capacity to modulate many functions of HASM; how the synthetic function of HASM, particularly through the release and expression of stem cell factor, has the potential to influence HMC number and activation in an extraordinarily potent and proinflammatory manner; and how these interactions between HMCs and HASM have potential consequences for airway structure and inflammation relevant to the disease process of asthma.

Store-operated calcium entry is required for sustained contraction and Ca2+ oscillations of airway smooth muscle.

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Chen, Jun; Sanderson, Michael J

2017-05-15

Airway hyper-responsiveness in asthma is driven by excessive contraction of airway smooth muscle cells (ASMCs). Agonist-induced Ca 2+ oscillations underlie this contraction of ASMCs and the magnitude of this contraction is proportional to the Ca 2+ oscillation frequency. Sustained contraction and Ca 2+ oscillations require an influx of extracellular Ca 2+ , although the mechanisms and pathways mediating this Ca 2+ influx during agonist-induced ASMC contraction are not well defined. By inhibiting store-operated calcium entry (SOCE) or voltage-gated Ca 2+ channels (VGCCs), we show that SOCE, rather than Ca 2+ influx via VGCCs, provides the major Ca 2+ entry pathway into ASMCs to sustain ASMCs contraction and Ca 2+ oscillations. SOCE may therefore serve as a potential target for new bronchodilators to reduce airway hyper-responsiveness in asthma. Asthma is characterized by airway hyper-responsiveness: the excessive contraction of airway smooth muscle. The extent of this airway contraction is proportional to the frequency of Ca 2+ oscillations within airway smooth muscle cells (ASMCs). Sustained Ca 2+ oscillations require a Ca 2+ influx to replenish Ca 2+ losses across the plasma membrane. Our previous studies implied store-operated calcium entry (SOCE) as the major pathway for this Ca 2+ influx. In the present study, we explore this hypothesis, by examining the effects of SOCE inhibitors (GSK7975A and GSK5498A) as well as L-type voltage-gated Ca 2+ channel inhibitors (nifedipine and nimodipine) on airway contraction and Ca 2+ oscillations and SOCE-mediated Ca 2+ influx in ASMCs within mouse precision-cut lung slices. We found that both GSK7975A and GSK5498A were able to fully relax methacholine-induced airway contraction by abolishing the Ca 2+ oscillations, in a manner similar to that observed in zero extracellular Ca 2+ ([Ca 2+ ] e ). In addition, GSK7975A and GSK5498A inhibited increases in intracellular Ca 2+ ([Ca 2+ ] i ) in ASMCs with depleted Ca 2+ -stores in

Roxithromycin inhibits VEGF-induced human airway smooth muscle cell proliferation: Opportunities for the treatment of asthma

International Nuclear Information System (INIS)

Pei, Qing-Mei; Jiang, Ping; Yang, Min; Qian, Xue-Jiao; Liu, Jiang-Bo; Kim, Sung-Ho

2016-01-01

Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodelling, which is associated with increased airway smooth muscle (ASM) mass. Roxithromycin (RXM) has been widely used in asthma treatment; however, its mechanism of action is poorly understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodelling in patients with asthma, and shown to promote ASM cell proliferation. Here, we investigated the effect of RXM on VEGF-induced ASM cell proliferation and attempted to elucidate the underlying mechanisms of action. We tested the effect of RXM on proliferation and cell cycle progression, as well as on the expression of phospho-VEGF receptor 2 (VEGFR2), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-Akt, and caveolin-1 in VEGF-stimulated ASM cells. RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. Additionally, VEGF-induced ASM cell proliferation was suppressed by inhibiting the activity of ERK1/2, but not that of Akt. Furthermore, RXM treatment inhibits VEGF-induced activation of VEGFR2 and ERK and downregulation of caveolin-1 in a dose-dependent manner. RXM also inhibited TGF-β-induced VEGF secretion by ASM cells and BEAS-2B cells. Collectively, our findings suggest that RXM inhibits VEGF-induced ASM cell proliferation by suppression of VEGFR2 and ERK1/2 activation and caveolin-1 down-regulation, which may be involved in airway remodelling. Further elucidation of the mechanisms underlying these observations should enable the development of treatments for smooth muscle hyperplasia-associated diseases of the airway such as asthma. - Highlights: • RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. • VEGF-induced cell proliferation was suppressed by inhibiting the activity of ERK1/2. • RXM inhibits activation of VEGFR2 and ERK and downregulation

Roxithromycin inhibits VEGF-induced human airway smooth muscle cell proliferation: Opportunities for the treatment of asthma

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Pei, Qing-Mei, E-mail: 34713316@qq.com [Department of Radiology, Tianjin Hospital of Integrated Traditional Chinese and Western Medicine, Tianjin (China); Jiang, Ping, E-mail: jiangping@163.com [Department of Respiration, Tianjin First Central Hospital, Tianjin (China); Yang, Min, E-mail: YangMin@163.com [Department of Respiration, Tianjin First Central Hospital, Tianjin (China); Qian, Xue-Jiao, E-mail: qianxuejiao@163.com [Department of Respiration, Tianjin First Central Hospital, Tianjin (China); Liu, Jiang-Bo, E-mail: LJB1984@163.com [Department of Respiration, Tianjin First Central Hospital, Tianjin (China); Kim, Sung-Ho, E-mail: chenghao0726@hotmail.com [Department of Respiration, Tianjin First Central Hospital, Tianjin (China)

2016-10-01

Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodelling, which is associated with increased airway smooth muscle (ASM) mass. Roxithromycin (RXM) has been widely used in asthma treatment; however, its mechanism of action is poorly understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodelling in patients with asthma, and shown to promote ASM cell proliferation. Here, we investigated the effect of RXM on VEGF-induced ASM cell proliferation and attempted to elucidate the underlying mechanisms of action. We tested the effect of RXM on proliferation and cell cycle progression, as well as on the expression of phospho-VEGF receptor 2 (VEGFR2), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-Akt, and caveolin-1 in VEGF-stimulated ASM cells. RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. Additionally, VEGF-induced ASM cell proliferation was suppressed by inhibiting the activity of ERK1/2, but not that of Akt. Furthermore, RXM treatment inhibits VEGF-induced activation of VEGFR2 and ERK and downregulation of caveolin-1 in a dose-dependent manner. RXM also inhibited TGF-β-induced VEGF secretion by ASM cells and BEAS-2B cells. Collectively, our findings suggest that RXM inhibits VEGF-induced ASM cell proliferation by suppression of VEGFR2 and ERK1/2 activation and caveolin-1 down-regulation, which may be involved in airway remodelling. Further elucidation of the mechanisms underlying these observations should enable the development of treatments for smooth muscle hyperplasia-associated diseases of the airway such as asthma. - Highlights: • RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. • VEGF-induced cell proliferation was suppressed by inhibiting the activity of ERK1/2. • RXM inhibits activation of VEGFR2 and ERK and downregulation

Role of dystrophin in airway smooth muscle phenotype, contraction and lung function.

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Pawan Sharma

Full Text Available Dystrophin links the transmembrane dystrophin-glycoprotein complex to the actin cytoskeleton. We have shown that dystrophin-glycoprotein complex subunits are markers for airway smooth muscle phenotype maturation and together with caveolin-1, play an important role in calcium homeostasis. We tested if dystrophin affects phenotype maturation, tracheal contraction and lung physiology. We used dystrophin deficient Golden Retriever dogs (GRMD and mdx mice vs healthy control animals in our approach. We found significant reduction of contractile protein markers: smooth muscle myosin heavy chain (smMHC and calponin and reduced Ca2+ response to contractile agonist in dystrophin deficient cells. Immunocytochemistry revealed reduced stress fibers and number of smMHC positive cells in dystrophin-deficient cells, when compared to control. Immunoblot analysis of Akt1, GSK3β and mTOR phosphorylation further revealed that downstream PI3K signaling, which is essential for phenotype maturation, was suppressed in dystrophin deficient cell cultures. Tracheal rings from mdx mice showed significant reduction in the isometric contraction to methacholine (MCh when compared to genetic control BL10ScSnJ mice (wild-type. In vivo lung function studies using a small animal ventilator revealed a significant reduction in peak airway resistance induced by maximum concentrations of inhaled MCh in mdx mice, while there was no change in other lung function parameters. These data show that the lack of dystrophin is associated with a concomitant suppression of ASM cell phenotype maturation in vitro, ASM contraction ex vivo and lung function in vivo, indicating that a linkage between the DGC and the actin cytoskeleton via dystrophin is a determinant of the phenotype and functional properties of ASM.

Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression.

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Woodruff, Prescott G; Dolganov, Gregory M; Ferrando, Ronald E; Donnelly, Samantha; Hays, Steven R; Solberg, Owen D; Carter, Roderick; Wong, Hofer H; Cadbury, Peggy S; Fahy, John V

2004-05-01

Bronchial hyperresponsiveness in mild to moderate asthma may result from airway smooth muscle cell proliferation or acquisition of a hypercontractile phenotype. Because these cells have not been well characterized in mild to moderate asthma, we examined the morphometric and gene expression characteristics of smooth muscle cells in this subgroup of patients with asthma. Using bronchial biopsies from 14 subjects with mild to moderate asthma and 15 control subjects, we quantified smooth muscle cell morphology by stereology and the expression of a panel of genes related to a hypercontractile phenotype of airway smooth muscle, using laser microdissection and two-step real-time polymerase chain reaction. We found that airway smooth muscle cell size was similar in both groups, but cell number was nearly twofold higher in subjects with asthma (p = 0.03), and the amount of smooth muscle in the submucosa was increased 50-83% (p 0.1). We conclude that airway smooth muscle proliferation is a pathologic characteristic of subjects with mild to moderate asthma. However, smooth muscle cells in mild to moderate asthma do not show hypertrophy or gene expression changes of a hypercontractile phenotype observed in vitro.

Cigarette Smoke and Estrogen Signaling in Human Airway Smooth Muscle

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Venkatachalem Sathish

2015-06-01

Full Text Available Aims: Cigarette smoke (CS in active smokers and second-hand smoke exposure exacerbate respiratory disorders such as asthma and chronic bronchitis. While women are known to experience a more asthmatic response to CS than emphysema in men, there is limited information on the mechanisms of CS-induced airway dysfunction. We hypothesize that CS interferes with a normal (protective bronchodilatory role of estrogens, thus worsening airway contractility. Methods: We tested effects of cigarette smoke extract (CSE on 17β-estradiol (E2 signaling in enzymatically-dissociated bronchial airway smooth muscle (ASM obtained from lung samples of non-smoking female patients undergoing thoracic surgery. Results: In fura-2 loaded ASM cells, CSE increased intracellular calcium ([Ca2+]i responses to 10µM histamine. Acute exposure to physiological concentrations of E2 decreased [Ca2+]i responses. However, in 24h exposed CSE cells, although expression of estrogen receptors was increased, the effect of E2 on [Ca2+]i was blunted. Acute E2 exposure also decreased store-operated Ca2+ entry and inhibited stromal interaction molecule 1 (STIM1 phosphorylation: effects blunted by CSE. Acute exposure to E2 increased cAMP, but less so in 24h CSE-exposed cells. 24h CSE exposure increased S-nitrosylation of ERα. Furthermore, 24h CSE-exposed bronchial rings showed increased bronchoconstrictor agonist responses that were not reduced as effectively by E2 compared to non-CSE controls. Conclusion: These data suggest that CS induces dysregulation of estrogen signaling in ASM, which could contribute to increased airway contractility in women exposed to CS.

PPARγ Ligands Regulate Noncontractile and Contractile Functions of Airway Smooth Muscle: Implications for Asthma Therapy

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Chantal Donovan

2012-01-01

Full Text Available In asthma, the increase in airway smooth muscle (ASM can contribute to inflammation, airway wall remodeling and airway hyperresponsiveness (AHR. Targetting peroxisome proliferator-activated receptor γ (PPARγ, a receptor upregulated in ASM in asthmatic airways, may provide a novel approach to regulate these contributions. This review summarises experimental evidence that PPARγ ligands, such as rosiglitazone (RGZ and pioglitazone (PGZ, inhibit proliferation and inflammatory cytokine production from ASM in vitro. In addition, inhaled administration of these ligands reduces inflammatory cell infiltration and airway remodelling in mouse models of allergen-induced airways disease. PPARγ ligands can also regulate ASM contractility, with acute treatment eliciting relaxation of mouse trachea in vitro through a PPARγ-independent mechanism. Chronic treatment can protect against the loss of bronchodilator sensitivity to β2-adrenoceptor agonists and inhibit the development of AHR associated with exposure to nicotine in utero or following allergen challenge. Of particular interest, a small clinical trial has shown that oral RGZ treatment improves lung function in smokers with asthma, a group that is generally unresponsive to conventional steroid treatment. These combined findings support further investigation of the potential for PPARγ agonists to target the noncontractile and contractile functions of ASM to improve outcomes for patients with poorly controlled asthma.

Contribution of SRF, Elk-1, and myocardin to airway smooth muscle remodeling in heaves, an asthma-like disease of horses.

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Chevigny, Mylène; Guérin-Montpetit, Karine; Vargas, Amandine; Lefebvre-Lavoie, Josiane; Lavoie, Jean-Pierre

2015-07-01

Myocyte hyperplasia and hypertrophy contribute to the increased mass of airway smooth muscle (ASM) in asthma. Serum-response factor (SRF) is a transcription factor that regulates myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with phosphorylated (p)Elk-1, it promotes ASM proliferation while binding to myocardin (MYOCD) leading to the expression of contractile elements in these tissues. The objective of this study was therefore to characterize the expression of SRF, pElk-1, and MYOCD in ASM cells from central and peripheral airways in heaves, a spontaneously occurring asthma-like disease of horses, and in controls. Six horses with heaves and five aged-matched controls kept in the same environment were studied. Nuclear protein expression of SRF, pElk-1, and MYOCD was evaluated in peripheral airways and endobronchial biopsies obtained during disease remission and after 1 and 30 days of naturally occurring antigenic exposure using immunohistochemistry and immunofluorescence techniques. Nuclear expression of SRF (P = 0.03, remission vs. 30 days) and MYOCD (P = 0.05, controls vs. heaves at 30 days) increased in the peripheral airways of horses with heaves during disease exacerbation, while MYOCD (P = 0.04, remission vs. 30 days) decreased in the central airways of control horses. No changes were observed in the expression of pElk-1 protein in either tissue. In conclusion, SRF and its cofactor MYOCD likely contribute to the hypertrophy of peripheral ASM observed in equine asthmatic airways, while the remodeling of the central airways is more static or involves different transcription factors. Copyright © 2015 the American Physiological Society.

Crosstalk between Gi and Gq/Gs pathways in airway smooth muscle regulates bronchial contractility and relaxation

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McGraw, Dennis W.; Elwing, Jean M.; Fogel, Kevin M.; Wang, Wayne C.H.; Glinka, Clare B.; Mihlbachler, Kathryn A.; Rothenberg, Marc E.; Liggett, Stephen B.

2007-01-01

Receptor-mediated airway smooth muscle (ASM) contraction via Gαq, and relaxation via Gαs, underlie the bronchospastic features of asthma and its treatment. Asthma models show increased ASM Gαi expression, considered the basis for the proasthmatic phenotypes of enhanced bronchial hyperreactivity to contraction mediated by M3-muscarinic receptors and diminished relaxation mediated by β2-adrenergic receptors (β2ARs). A causal effect between Gi expression and phenotype has not been established, n...

Inhaled corticosteroids inhibit substance P receptor expression in asthmatic rat airway smooth muscle cells

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Li Miao

2012-12-01

Full Text Available Abstract Background Neurokinins (NKs participate in asthmatic airway inflammation, but the effects of NKs on airway smooth muscle cells (ASMCs and those of corticosteroids on NKs are unknown. Methods To investigate the effect of budesonide on substance P (NK-1 receptor (NK-1R expression in the lung and ASMCs, 45 Wistar rats were randomly divided into three groups: control, asthmatic, and budesonide treatment. Aerosolized ovalbumin was used to generate the asthmatic rat model, and budesonide was administered after ovalbumin inhalation. On day 21, bronchial responsiveness tests, bronchoalveolar lavage, and cell counting were conducted. NK-1R protein expression in the lung was investigated by immunohistochemistry and image analysis. Primary rat ASMC cultures were established, and purified ASMCs of the fourth passage were collected for mRNA and protein studies via real-time RT-PCR, immunocytochemistry, and image analysis. Results NK-1R mRNA and protein expression in the budesonide treatment group rat’s lung and ASMCs were less than that in the asthmatic group but greater than that in the control group. Conclusions NK-1R is involved in the pathogenesis of asthma and that budesonide may downregulate the expression of NK-1R in the ASMCs and airways of asthmatic rats, which may alleviate neurogenic airway inflammation.

Matrix stiffness-modulated proliferation and secretory function of the airway smooth muscle cells.

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Shkumatov, Artem; Thompson, Michael; Choi, Kyoung M; Sicard, Delphine; Baek, Kwanghyun; Kim, Dong Hyun; Tschumperlin, Daniel J; Prakash, Y S; Kong, Hyunjoon

2015-06-01

Multiple pulmonary conditions are characterized by an abnormal misbalance between various tissue components, for example, an increase in the fibrous connective tissue and loss/increase in extracellular matrix proteins (ECM). Such tissue remodeling may adversely impact physiological function of airway smooth muscle cells (ASMCs) responsible for contraction of airways and release of a variety of bioactive molecules. However, few efforts have been made to understand the potentially significant impact of tissue remodeling on ASMCs. Therefore, this study reports how ASMCs respond to a change in mechanical stiffness of a matrix, to which ASMCs adhere because mechanical stiffness of the remodeled airways is often different from the physiological stiffness. Accordingly, using atomic force microscopy (AFM) measurements, we found that the elastic modulus of the mouse bronchus has an arithmetic mean of 23.1 ± 14 kPa (SD) (median 18.6 kPa). By culturing ASMCs on collagen-conjugated polyacrylamide hydrogels with controlled elastic moduli, we found that gels designed to be softer than average airway tissue significantly increased cellular secretion of vascular endothelial growth factor (VEGF). Conversely, gels stiffer than average airways stimulated cell proliferation, while reducing VEGF secretion and agonist-induced calcium responses of ASMCs. These dependencies of cellular activities on elastic modulus of the gel were correlated with changes in the expression of integrin-β1 and integrin-linked kinase (ILK). Overall, the results of this study demonstrate that changes in matrix mechanics alter cell proliferation, calcium signaling, and proangiogenic functions in ASMCs. Copyright © 2015 the American Physiological Society.

Thiazolidinediones inhibit airway smooth muscle release of the chemokine CXCL10: in vitro comparison with current asthma therapies

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Seidel Petra; Alkhouri Hatem; Lalor Daniel J; Burgess Janette K; Armour Carol L; Hughes J

2012-01-01

Abstract Background Activated mast cells are present within airway smooth muscle (ASM) bundles in eosinophilic asthma. ASM production of the chemokine CXCL10 plays a role in their recruitment. Thus the effects of glucocorticoids (fluticasone, budesonide), long-acting β2-agonists (salmeterol, formoterol) and thiazolidinediones (ciglitazone, rosiglitazone) on CXCL10 production by ASM cells (ASMC) from people with and without asthma were investigated in vitro. Methods Confluent serum-deprived ce...

Regulation of GPCR-mediated smooth muscle contraction : implications for asthma and pulmonary hypertension

NARCIS (Netherlands)

Wright, D B; Tripathi, S; Sikarwar, A; Santosh, K T; Perez-Zoghbi, J; Ojo, O O; Irechukwu, N; Ward, J P T; Schaafsma, D

Contractile G-protein-coupled receptors (GPCRs) have emerged as key regulators of smooth muscle contraction, both under healthy and diseased conditions. This brief review will discuss some key topics and novel insights regarding GPCR-mediated airway and vascular smooth muscle contraction as

Effects of acute ethanol exposure on cytokine production by primary airway smooth muscle cells

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Kaphalia, Lata; Kalita, Mridul [Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX (United States); Kaphalia, Bhupendra S. [Department of Pathology, University of Texas Medical Branch, Galveston, TX (United States); Calhoun, William J., E-mail: William.Calhoun@utmb.edu [Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX (United States)

2016-02-01

Both chronic and binge alcohol abuse can be significant risk factors for inflammatory lung diseases such as acute respiratory distress syndrome and chronic obstructive pulmonary disease. However, metabolic basis of alcohol-related lung disease is not well defined, and may include key metabolites of ethanol [EtOH] in addition to EtOH itself. Therefore, we investigated the effects of EtOH, acetaldehyde [ACE], and fatty acid ethyl esters [FAEEs] on oxidative stress, endoplasmic reticulum (ER) stress, AMP-activated protein kinase (AMPK) signaling and nuclear translocation of phosphorylated (p)-NF-κB p65 in primary human airway smooth muscle (HASM) cells stimulated to produce cytokines using LPS exposure. Both FAEEs and ACE induced evidence of cellular oxidative stress and ER stress, and increased p-NF-κB in nuclear extracts. EtOH and its metabolites decreased p-AMPKα activation, and induced expression of fatty acid synthase, and decreased expression of sirtuin 1. In general, EtOH decreased secretion of IP-10, IL-6, eotaxin, GCSF, and MCP-1. However, FAEEs and ACE increased these cytokines, suggesting that both FAEEs and ACE as compared to EtOH itself are proinflammatory. A direct effect of EtOH could be consistent with blunted immune response. Collectively, these two features of EtOH exposure, coupled with the known inhibition of innate immune response in our model might explain some clinical manifestations of EtOH exposure in the lung. - Highlights: • Metabolic basis for EtOH toxicity was studied in human airway smooth muscle (HASM) cells. • In HASM cells, EtOH metabolites were found to be relatively more toxic than EtOH itself. • EtOH metabolites mediate deactivation of AMPK via oxidative stress and ER stress. • EtOH metabolites were found to be more proinflammatory than EtOH itself in HASM cells.

Effects of acute ethanol exposure on cytokine production by primary airway smooth muscle cells

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Kaphalia, Lata; Kalita, Mridul; Kaphalia, Bhupendra S.; Calhoun, William J.

2016-01-01

Both chronic and binge alcohol abuse can be significant risk factors for inflammatory lung diseases such as acute respiratory distress syndrome and chronic obstructive pulmonary disease. However, metabolic basis of alcohol-related lung disease is not well defined, and may include key metabolites of ethanol [EtOH] in addition to EtOH itself. Therefore, we investigated the effects of EtOH, acetaldehyde [ACE], and fatty acid ethyl esters [FAEEs] on oxidative stress, endoplasmic reticulum (ER) stress, AMP-activated protein kinase (AMPK) signaling and nuclear translocation of phosphorylated (p)-NF-κB p65 in primary human airway smooth muscle (HASM) cells stimulated to produce cytokines using LPS exposure. Both FAEEs and ACE induced evidence of cellular oxidative stress and ER stress, and increased p-NF-κB in nuclear extracts. EtOH and its metabolites decreased p-AMPKα activation, and induced expression of fatty acid synthase, and decreased expression of sirtuin 1. In general, EtOH decreased secretion of IP-10, IL-6, eotaxin, GCSF, and MCP-1. However, FAEEs and ACE increased these cytokines, suggesting that both FAEEs and ACE as compared to EtOH itself are proinflammatory. A direct effect of EtOH could be consistent with blunted immune response. Collectively, these two features of EtOH exposure, coupled with the known inhibition of innate immune response in our model might explain some clinical manifestations of EtOH exposure in the lung. - Highlights: • Metabolic basis for EtOH toxicity was studied in human airway smooth muscle (HASM) cells. • In HASM cells, EtOH metabolites were found to be relatively more toxic than EtOH itself. • EtOH metabolites mediate deactivation of AMPK via oxidative stress and ER stress. • EtOH metabolites were found to be more proinflammatory than EtOH itself in HASM cells.

The effects of interleukin-8 on airway smooth muscle contraction in cystic fibrosis

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Safka Katherine

2008-12-01

Full Text Available Abstract Background Many cystic fibrosis (CF patients display airway hyperresponsiveness and have symptoms of asthma such as cough, wheezing and reversible airway obstruction. Chronic airway bacterial colonization, associated with neutrophilic inflammation and high levels of interleukin-8 (IL-8 is also a common occurrence in these patients. The aim of this work was to determine the responsiveness of airway smooth muscle to IL-8 in CF patients compared to non-CF individuals. Methods Experiments were conducted on cultured ASM cells harvested from subjects with and without CF (control subjects. Cells from the 2nd to 5th passage were studied. Expression of the IL-8 receptors CXCR1 and CXCR2 was assessed by flow cytometry. The cell response to IL-8 was determined by measuring intracellular calcium concentration ([Ca2+]i, cell contraction, migration and proliferation. Results The IL-8 receptors CXCR1 and CXCR2 were expressed in both non-CF and CF ASM cells to a comparable extent. IL-8 (100 nM induced a peak Ca2+ release that was higher in control than in CF cells: 228 ± 7 versus 198 ± 10 nM (p 20 in CF than in control cells. In addition, MLC20 expression was also increased in CF cells. Exposure to IL-8 induced migration and proliferation of both groups of ASM cells but was not different between CF and non-CF cells. Conclusion ASM cells of CF patients are more contractile to IL-8 than non-CF ASM cells. This enhanced contractility may be due to an increase in the amount of contractile protein MLC20. Higher expression of MLC20 by CF cells could contribute to airway hyperresponsiveness to IL-8 in CF patients.

Targeting Phosphoinositide 3-Kinase γ in Airway Smooth Muscle Cells to Suppress Interleukin-13-Induced Mouse Airway Hyperresponsiveness

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Jiang, Haihong; Xie, Yan; Abel, Peter W.; Toews, Myron L.; Townley, Robert G.; Casale, Thomas B.

2012-01-01

We recently reported that phosphoinositide 3-kinase γ (PI3Kγ) directly regulates airway smooth muscle (ASM) contraction by modulating Ca2+ oscillations. Because ASM contraction plays a critical role in airway hyperresponsiveness (AHR) of asthma, the aim of the present study was to determine whether targeting PI3Kγ in ASM cells could suppress AHR in vitro and in vivo. Intranasal administration into mice of interleukin-13 (IL-13; 10 μg per mouse), a key pathophysiologic cytokine in asthma, induced AHR after 48 h, as assessed by invasive tracheostomy. Intranasal administration of a broad-spectrum PI3K inhibitor or a PI3Kγ-specific inhibitor 1 h before AHR assessment attenuated IL-13 effects. Airway responsiveness to bronchoconstrictor agonists was also examined in precision-cut mouse lung slices pretreated without or with IL-13 for 24 h. Acetylcholine and serotonin dose-response curves indicated that IL-13-treated lung slices had a 40 to 50% larger maximal airway constriction compared with controls. Furthermore, acetylcholine induced a larger initial Ca2+ transient and increased Ca2+ oscillations in IL-13-treated primary mouse ASM cells compared with control cells, correlating with increased cell contraction. As expected, PI3Kγ inhibitor treatment attenuated IL-13-augmented airway contractility of lung slices and ASM cell contraction. In both control and IL-13-treated ASM cells, small interfering RNA-mediated knockdown of PI3Kγ by 70% only reduced the initial Ca2+ transient by 20 to 30% but markedly attenuated Ca2+ oscillations and contractility of ASM cells by 50 to 60%. This report is the first to demonstrate that PI3Kγ in ASM cells is important for IL-13-induced AHR and that acute treatment with a PI3Kγ inhibitor can ameliorate AHR in a murine model of asthma. PMID:22543031

Protective effects of anisodamine on cigarette smoke extract-induced airway smooth muscle cell proliferation and tracheal contractility

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Xu, Guang-Ni; Yang, Kai; Xu, Zu-Peng; Zhu, Liang; Hou, Li-Na; Qi, Hong; Chen, Hong-Zhuan; Cui, Yong-Yao

2012-01-01

Anisodamine, an antagonist of muscarinic acetylcholine receptors (mAChRs), has been used therapeutically to improve smooth muscle function, including microvascular, intestinal and airway spasms. Our previous studies have revealed that airway hyper-reactivity could be prevented by anisodamine. However, whether anisodamine prevents smoking-induced airway smooth muscle (ASM) cell proliferation remained unclear. In this study, a primary culture of rat ASM cells was used to evaluate an ASM phenotype through the ability of the cells to proliferate and express contractile proteins in response to cigarette smoke extract (CSE) and intervention of anisodamine. Our results showed that CSE resulted in an increase in cyclin D1 expression concomitant with the G0/G1-to-S phase transition, and high expression of M2 and M3. Functional studies showed that tracheal hyper-contractility accompanied contractile marker α-SMA high-expression. These changes, which occur only after CSE stimulation, were prevented and reversed by anisodamine, and CSE-induced cyclin D1 expression was significantly inhibited by anisodamine and the specific inhibitor U0126, BAY11-7082 and LY294002. Thus, we concluded that the protective and reversal effects and mechanism of anisodamine on CSE-induced events might involve, at least partially, the ERK, Akt and NF-κB signaling pathways associated with cyclin D1 via mAChRs. Our study validated that anisodamine intervention on ASM cells may contribute to anti-remodeling properties other than bronchodilation. -- Highlights: ► CSE induces tracheal cell proliferation, hyper-contractility and α-SMA expression. ► Anisodamine reverses CSE-induced tracheal hyper-contractility and cell proliferation. ► ERK, PI3K, and NF-κB pathways and cyclin D1 contribute to the reversal effect.

Effects of Ginger and Its Constituents on Airway Smooth Muscle Relaxation and Calcium Regulation

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Siviski, Matthew E.; Zhang, Yi; Xu, Carrie; Hoonjan, Bhupinder; Emala, Charles W.

2013-01-01

The prevalence of asthma has increased in recent years, and is characterized by airway hyperresponsiveness and inflammation. Many patients report using alternative therapies to self-treat asthma symptoms as adjuncts to short-acting and long-acting β-agonists and inhaled corticosteroids (ICS). As many as 40% of patients with asthma use herbal therapies to manage asthma symptoms, often without proven efficacy or known mechanisms of action. Therefore, investigations of both the therapeutic and possible detrimental effects of isolated components of herbal treatments on the airway are important. We hypothesized that ginger and its active components induce bronchodilation by modulating intracellular calcium ([Ca2+]i) in airway smooth muscle (ASM). In isolated human ASM, ginger caused significant and rapid relaxation. Four purified constituents of ginger were subsequently tested for ASM relaxant properties in both guinea pig and human tracheas: [6]-gingerol, [8]-gingerol, and [6]-shogaol induced rapid relaxation of precontracted ASM (100–300 μM), whereas [10]-gingerol failed to induce relaxation. In human ASM cells, exposure to [6]-gingerol, [8]-gingerol, and [6]-shogaol, but not [10]-gingerol (100 μM), blunted subsequent Ca2+ responses to bradykinin (10 μM) and S-(−)-Bay K 8644 (10 μM). In A/J mice, the nebulization of [8]-gingerol (100 μM), 15 minutes before methacholine challenge, significantly attenuated airway resistance, compared with vehicle. Taken together, these novel data show that ginger and its isolated active components, [6]-gingerol, [8]-gingerol, and [6]-shogaol, relax ASM, and [8]-gingerol attenuates airway hyperresponsiveness, in part by altering [Ca2+]i regulation. These purified compounds may provide a therapeutic option alone or in combination with accepted therapeutics, including β2-agonists, in airway diseases such as asthma. PMID:23065130

Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone.

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Pearson, Helen; Britt, Rodney D; Pabelick, Christine M; Prakash, Y S; Amrani, Yassine; Pandya, Hitesh C

2015-12-01

Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. Cultured fetal human ASM cells stimulated with TNF-α (0-20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases.

The Oligo Fucoidan Inhibits Platelet-Derived Growth Factor-Stimulated Proliferation of Airway Smooth Muscle Cells

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Chao-Huei Yang

2016-01-01

Full Text Available In the pathogenesis of asthma, the proliferation of airway smooth muscle cells (ASMCs is a key factor in airway remodeling and causes airway narrowing. In addition, ASMCs are also the effector cells of airway inflammation. Fucoidan extracted from marine brown algae polysaccharides has antiviral, antioxidant, antimicrobial, anticlotting, and anticancer properties; however, its effectiveness for asthma has not been elucidated thus far. Platelet-derived growth factor (PDGF-treated primary ASMCs were cultured with or without oligo-fucoidan (100, 500, or 1000 µg/mL to evaluate its effects on cell proliferation, cell cycle, apoptosis, and Akt, ERK1/2 signaling pathway. We found that PDGF (40 ng/mL increased the proliferation of ASMCs by 2.5-fold after 48 h (p < 0.05. Oligo-fucoidan reduced the proliferation of PDGF-stimulated ASMCs by 75%–99% after 48 h (p < 0.05 and induced G1/G0 cell cycle arrest, but did not induce apoptosis. Further, oligo-fucoidan supplementation reduced PDGF-stimulated extracellular signal-regulated kinase (ERK1/2, Akt, and nuclear factor (NF-κB phosphorylation. Taken together, oligo-fucoidan supplementation might reduce proliferation of PDGF-treated ASMCs through the suppression of ERK1/2 and Akt phosphorylation and NF-κB activation. The results provide basis for future animal experiments and human trials.

Endobronchial Ultrasound Reliably Quantifies Airway Smooth Muscle Remodeling in an Equine Asthma Model.

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Michela Bullone

Full Text Available Endobronchial ultrasonography (EBUS revealed differences in the thickness of the layer representing subepithelial tissues (L2 between human asthmatics and controls, but whether this measurement correlates with airway smooth muscle (ASM remodeling in asthma is unknown. In this study, we sought to determine the ability of EBUS to predict histological ASM remodeling in normal and equine asthmatic airways. We studied 109 isolated bronchi from the lungs of 13 horses. They underwent EBUS examination using a 30 MHz radial probe before being processed for histology. ASM remodeling parameters were evaluated in EBUS images (L2 thickness, L2 area, L2 area/internal perimeter [Pi] and L2 area/Pi2 and histological cuts (ASM area/Pi2, and compared. EBUS was then performed ex vivo on the lungs of 4 horses with heaves, an asthma-like condition of horses, and 7 controls to determine whether central bronchial remodeling could be detected with this technique. An optimized approach was developed based on data variability within airways, subjects, and groups, and then validated in 7 horses (3 controls, 4 with heaves that underwent EBUS in vivo. L2 area was significantly associated to ASM area in isolated lungs (p<0.0001, in the absence of significant bias related to the airway size. Bronchial size significantly affected EBUS ASM-related parameters, except for L2 area/Pi2. L2 area/Pi2 was increased in the airways of asthmatic horses compared to controls, both ex vivo and in vivo (p<0.05. Bronchial histology confirmed our findings (AASM/Pi2 was increased in asthmatic horses compared to controls, p<0.05. In both horses with heaves and controls, L2 was composed of ASM for the outer 75% of its thickness and by ECM for the remaining inner 25%. In conclusion, EBUS reliably allows assessment of asthma-associated ASM remodeling of central airways in a non-invasive way.

Reverse mode Na+/Ca2+ exchange mediated by STIM1 contributes to Ca2+ influx in airway smooth muscle following agonist stimulation

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Fox Jane

2010-12-01

Full Text Available Abstract Background Agonist stimulation of airway smooth muscle (ASM results in IP3 mediated Ca2+ release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na+ influx. This localised increase in Na+ levels can potentially switch the Na+/Ca2+ exchanger into reverse mode and so result in a further influx of Ca2+. The aim of this study was to characterise the expression and physiological function of the Na+/Ca2+ exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca2+ influx into these cells. Methods The expression profile of NCX (which encodes the Na+/Ca2+ exchanger homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca2+ measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX and target specific siRNA were utilised as tools to inhibit NCX function. Results NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na+ loading and addition of extracellular Ca2+ induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1 and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca2+ and induced relaxation of porcine

Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels.

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Montaño, Luis M; Cruz-Valderrama, José E; Figueroa, Alejandra; Flores-Soto, Edgar; García-Hernández, Luz M; Carbajal, Verónica; Segura, Patricia; Méndez, Carmen; Díaz, Verónica; Barajas-López, Carlos

2011-10-01

In airway smooth muscle (ASM), adenosine 5'-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K(+) currents (I (K)) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,β-methylene-ATP was inactive from 1 to 100 μM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I (K) increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I (K) was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I (K) increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y(2) and P2Y(4) receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y(4) and P2Y(2) receptors; (2) induces I (K) increment through activation of the delayed rectifier K(+) channels and the high-conductance Ca(2+)-dependent K(+) channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I (K) increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.

Acrolein relaxes mouse isolated tracheal smooth muscle via a TRPA1-dependent mechanism.

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Cheah, Esther Y; Burcham, Philip C; Mann, Tracy S; Henry, Peter J

2014-05-01

Airway sensory C-fibres express TRPA1 channels which have recently been identified as a key chemosensory receptor for acrolein, a toxic and highly prevalent component of smoke. TRPA1 likely plays an intermediary role in eliciting a range of effects induced by acrolein including cough and neurogenic inflammation. Currently, it is not known whether acrolein-induced activation of TRPA1 produces other airway effects including relaxation of mouse airway smooth muscle. The aims of this study were to examine the effects of acrolein on airway smooth muscle tone in mouse isolated trachea, and to characterise the cellular and molecular mechanisms underpinning the effects of acrolein. Isometric tension recording studies were conducted on mouse isolated tracheal segments to characterise acrolein-induced relaxation responses. Release of the relaxant PGE₂ was measured by EIA to examine its role in the response. Use of selective antagonists/inhibitors permitted pharmacological characterisation of the molecular and cellular mechanisms underlying this relaxation response. Acrolein induced dose-dependent relaxation responses in mouse isolated tracheal segments. Importantly, these relaxation responses were significantly inhibited by the TRPA1 antagonists AP-18 and HC-030031, an NK₁ receptor antagonist RP-67580, and the EP₂ receptor antagonist PF-04418948, whilst completely abolished by the non-selective COX inhibitor indomethacin. Acrolein also caused rapid PGE₂ release which was suppressed by HC-030031. In summary, acrolein induced a novel bronchodilator response in mouse airways. Pharmacologic studies indicate that acrolein-induced relaxation likely involves interplay between TRPA1-expressing airway sensory C-fibres, NK₁ receptor-expressing epithelial cells, and EP₂-receptor expressing airway smooth muscle cells. Copyright © 2014 Elsevier Inc. All rights reserved.

The Expression of NOX4 in Smooth Muscles of Small Airway Correlates with the Disease Severity of COPD.

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Liu, Xianyan; Hao, Binwei; Ma, Ailing; He, Jinxi; Liu, Xiaoming; Chen, Juan

Airway smooth muscle (ASM) remodeling is a hallmark in chronic obstructive pulmonary disease (COPD), and nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases (NOXs) produced reactive oxygen species (ROS) play a crucial role in COPD pathogenesis. In the present study, the expression of NOX4 and its correlation with the ASM hypertrophy/hyperplasia, clinical pulmonary functions, and the expression of transforming growth factor β (TGF- β ) in the ASM of COPD small airways were investigated by semiquantitative morphological and/or immunohistochemistry staining methods. The results showed that an elevated expression of NOX4 and TGF- β , along with an increased volume of ASM mass, was found in the ASM of small airways in COPD patients. The abundance of NOX4 protein in the ASM was increased with disease severity and inversely correlated with the pulmonary functions in COPD patients. In addition, the expression of NOX4 and ASM marker α -SMA was colocalized, and the increased NOX4 expression was found to accompany an upregulated expression of TGF- β in the ASM of small airways of COPD lung. These results indicate that NOX4 may be a key regulator in ASM remodeling of small airway, in part through a mechanism interacting with TGF- β signaling in the pathogenesis of COPD, which warrants further investigation.

Differential expression of peroxisome proliferator activated receptor gamma and cyclin D1 does not affect proliferation of asthma- and non-asthma-derived airway smooth muscle cells

NARCIS (Netherlands)

Lau, Justine Y; Oliver, Brian G; Moir, Lyn M; Black, Judith L; Burgess, Janette K

UNLABELLED: PPARgamma levels in asthma- and non-asthma-derived airway smooth muscle cells and PPARgamma activation-induced cell proliferation were investigated. In the presence of FBS, PPARgamma levels were higher in subconfluent asthma-derived cells but lower in confluent cells compared with

Orosomucoid-like 3 (ORMDL3) upregulates airway smooth muscle proliferation, contraction, and Ca2+ oscillations in asthma.

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Chen, Jun; Miller, Marina; Unno, Hirotoshi; Rosenthal, Peter; Sanderson, Michael J; Broide, David H

2017-09-07

Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3 Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3 Zp3-Cre mice, which do not have a blood supply. Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3 Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca 2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Th1 cytokine-induced syndecan-4 shedding by airway smooth muscle cells is dependent on mitogen-activated protein kinases.

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Tan, Xiahui; Khalil, Najwa; Tesarik, Candice; Vanapalli, Karunasri; Yaputra, Viki; Alkhouri, Hatem; Oliver, Brian G G; Armour, Carol L; Hughes, J Margaret

2012-04-01

In asthma, airway smooth muscle (ASM) chemokine secretion can induce mast cell recruitment into the airways. The functions of the mast cell chemoattractant CXCL10, and other chemokines, are regulated by binding to heparan sulphates such as syndecan-4. This study is the first demonstration that airway smooth muscle cells (ASMC) from people with and without asthma express and shed syndecan-4 under basal conditions. Syndecan-4 shedding was enhanced by stimulation for 24 h with the Th1 cytokines interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α), but not interferon-γ (IFNγ), nor the Th2 cytokines IL-4 and IL-13. ASMC stimulation with IL-1β, TNF-α, and IFNγ (cytomix) induced the highest level of syndecan-4 shedding. Nonasthmatic and asthmatic ASM cell-associated syndecan-4 protein expression was also increased by TNF-α or cytomix at 4-8 h, with the highest levels detected in cytomix-stimulated asthmatic cells. Cell-associated syndecan-4 levels were decreased by 24 h, whereas shedding remained elevated at 24 h, consistent with newly synthesized syndecan-4 being shed. Inhibition of ASMC matrix metalloproteinase-2 did not prevent syndecan-4 shedding, whereas inhibition of ERK MAPK activation reduced shedding from cytomix-stimulated ASMC. Although ERK inhibition had no effect on syndecan-4 mRNA levels stimulated by cytomix, it did cause an increase in cell-associated syndecan-4 levels, consistent with the shedding being inhibited. In conclusion, ASMC produce and shed syndecan-4 and although this is increased by the Th1 cytokines, the MAPK ERK only regulates shedding. ASMC syndecan-4 production during Th1 inflammatory conditions may regulate chemokine activity and mast cell recruitment to the ASM in asthma.

Stress and strain in the contractile and cytoskeletal filaments of airway smooth muscle.

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Deng, Linhong; Bosse, Ynuk; Brown, Nathan; Chin, Leslie Y M; Connolly, Sarah C; Fairbank, Nigel J; King, Greg G; Maksym, Geoffrey N; Paré, Peter D; Seow, Chun Y; Stephen, Newman L

2009-10-01

Stress and strain are omnipresent in the lung due to constant lung volume fluctuation associated with respiration, and they modulate the phenotype and function of all cells residing in the airways including the airway smooth muscle (ASM) cell. There is ample evidence that the ASM cell is very sensitive to its physical environment, and can alter its structure and/or function accordingly, resulting in either desired or undesired consequences. The forces that are either conferred to the ASM cell due to external stretching or generated inside the cell must be borne and transmitted inside the cytoskeleton (CSK). Thus, maintaining appropriate levels of stress and strain within the CSK is essential for maintaining normal function. Despite the importance, the mechanisms regulating/dysregulating ASM cytoskeletal filaments in response to stress and strain remained poorly understood until only recently. For example, it is now understood that ASM length and force are dynamically regulated, and both can adapt over a wide range of length, rendering ASM one of the most malleable living tissues. The malleability reflects the CSK's dynamic mechanical properties and plasticity, both of which strongly interact with the loading on the CSK, and all together ultimately determines airway narrowing in pathology. Here we review the latest advances in our understanding of stress and strain in ASM cells, including the organization of contractile and cytoskeletal filaments, range and adaptation of functional length, structural and functional changes of the cell in response to mechanical perturbation, ASM tone as a mediator of strain-induced responses, and the novel glassy dynamic behaviors of the CSK in relation to asthma pathophysiology.

Exploiting the relationship between birefringence and force to measure airway smooth muscle contraction with PS-OCT (Conference Presentation)

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Adams, David C.; Hariri, Lida P.; Holz, Jasmin A.; Szabari, Margit V.; Harris, R. Scott; Cho, Jocelyn L.; Hamilos, Daniel L.; Luster, Andrew D.; Medoff, Benjamin D.; Suter, Melissa J.

2016-03-01

The ability to observe airway dynamics is fundamental to forming a complete understanding of pulmonary diseases such as asthma. We have previously demonstrated that Optical Coherence Tomography (OCT) can be used to observe structural changes in the airway during bronchoconstriction, but standard OCT lacks the contrast to discriminate airway smooth muscle (ASM) bands- ASM being responsible for generating the force that drives airway constriction- from the surrounding tissue. Since ASM in general exhibits a greater degree of birefringence than the surrounding tissue, a potential solution to this problem lies in the implementation of polarization sensitivity (PS) to the OCT system. By modifying the OCT system so that it is sensitive to the birefringence of tissue under inspection, we can visualize the ASM with much greater clarity and definition. In this presentation we show that the force of contraction can be indirectly measured by an associated increase in the birefringence signal of the ASM. We validate this approach by attaching segments of swine trachea to an isometric force transducer and stimulating contraction, while simultaneously measuring the exerted force and imaging the segment with PS-OCT. We then show how our results may be used to extrapolate the force of contraction of closed airways in absence of additional measurement devices. We apply this technique to assess ASM contractility volumetrically and in vivo, in both asthmatic and non-asthmatic human volunteers.

Bitter taste receptors in the wrong place: novel airway smooth muscle targets for treating asthma.

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Liggett, Stephen B

2014-01-01

There is a need to expand the classes of drugs used to treat obstructive lung diseases to achieve better outcomes. With only one class of direct bronchodilators (β-agonists), we sought to find receptors on human airway smooth muscle (ASM) that act via a unique mechanism to relax the muscle, have a diverse agonist binding profile to enhance the probability of finding new therapeutics, and relax ASM with equal or greater efficacy than β-agonists. We have found that human and mouse ASM express six bitter taste receptor (TAS2R) subtypes, previously thought only to exist in taste buds of the tongue. Agonists acting at TAS2Rs evoke profound bronchodilation via a Ca(2+)-dependent mechanism. TAS2R function is not altered in asthma models, undergoes minimal tachyphylaxis upon repetitive dosing, and relaxes even under extreme desensitization of relaxation by β-agonists. Taken together, TAS2Rs on ASM represent a novel pathway to consider for development of agonists in the treatment of asthma and chronic obstructive lung disease.

Cyclic mechanical strain-induced proliferation and migration of human airway smooth muscle cells: role of EMMPRIN and MMPs.

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Hasaneen, Nadia A; Zucker, Stanley; Cao, Jian; Chiarelli, Christian; Panettieri, Reynold A; Foda, Hussein D

2005-09-01

Airway smooth muscle (ASM) proliferation and migration are major components of airway remodeling in asthma. Asthmatic airways are exposed to mechanical strain, which contributes to their remodeling. Matrix metalloproteinase (MMP) plays an important role in remodeling. In the present study, we examined if the mechanical strain of human ASM (HASM) cells contributes to their proliferation and migration and the role of MMPs in this process. HASM were exposed to mechanical strain using the FlexCell system. HASM cell proliferation, migration and MMP release, activation, and expression were assessed. Our results show that cyclic strain increased the proliferation and migration of HASM; cyclic strain increased release and activation of MMP-1, -2, and -3 and membrane type 1-MMP; MMP release was preceded by an increase in extracellular MMP inducer; Prinomastat [a MMP inhibitor (MMPI)] significantly decreased cyclic strain-induced proliferation and migration of HASM; and the strain-induced increase in the release of MMPs was accompanied by an increase in tenascin-C release. In conclusion, cyclic mechanical strain plays an important role in HASM cell proliferation and migration. This increase in proliferation and migration is through an increase in MMP release and activation. Pharmacological MMPIs should be considered in the pursuit of therapeutic options for airway remodeling in asthma.

Major vault protein in cardiac and smooth muscle.

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Shults, Nataliia V; Das, Dividutta; Suzuki, Yuichiro J

Major vault protein (MVP) is the major component of the vault particle whose functions are not well understood. One proposed function of the vault is to serve as a mechanism of drug transport, which confers drug resistance in cancer cells. We show that MVP can be found in cardiac and smooth muscle. In human airway smooth muscle cells, knocking down MVP was found to cause cell death, suggesting that MVP serves as a cell survival factor. Further, our laboratory found that MVP is S-glutathionylated in response to ligand/receptor-mediated cell signaling. The S-glutathionylation of MVP appears to regulate protein-protein interactions between MVP and a protein called myosin heavy chain 9 (MYH9). Through MYH9 and Vsp34, MVP may form a complex with Beclin-1 that regulates autophagic cell death. In pulmonary vascular smooth muscle, proteasome inhibition promotes the ubiquitination of MVP, which may function as a mechanism of proteasome inhibition-mediated cell death. Investigating the functions and the regulatory mechanisms of MVP and vault particles is an exciting new area of research in cardiovascular/pulmonary pathophysiology.

MicroRNA Mediated Chemokine Responses in Human Airway Smooth Muscle Cells.

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Mythili Dileepan

Full Text Available Airway smooth muscle (ASM cells play a critical role in the pathophysiology of asthma due to their hypercontractility and their ability to proliferate and secrete inflammatory mediators. microRNAs (miRNAs are gene regulators that control many signaling pathways and thus serve as potential therapeutic alternatives for many diseases. We have previously shown that miR-708 and miR-140-3p regulate the MAPK and PI3K signaling pathways in human ASM (HASM cells following TNF-α exposure. In this study, we investigated the regulatory effect of these miRNAs on other asthma-related genes. Microarray analysis using the Illumina platform was performed with total RNA extracted from miR-708 (or control miR-transfected HASM cells. Inhibition of candidate inflammation-associated gene expression was further validated by qPCR and ELISA. The most significant biologic functions for the differentially expressed gene set included decreased inflammatory response, cytokine expression and signaling. qPCR revealed inhibition of expression of CCL11, CXCL10, CCL2 and CXCL8, while the release of CCL11 was inhibited in miR-708-transfected cells. Transfection of cells with miR-140-3p resulted in inhibition of expression of CCL11, CXCL12, CXCL10, CCL5 and CXCL8 and of TNF-α-induced CXCL12 release. In addition, expression of RARRES2, CD44 and ADAM33, genes known to contribute to the pathophysiology of asthma, were found to be inhibited in miR-708-transfected cells. These results demonstrate that miR-708 and miR-140-3p exert distinct effects on inflammation-associated gene expression and biological function of ASM cells. Targeting these miRNA networks may provide a novel therapeutic mechanism to down-regulate airway inflammation and ASM proliferation in asthma.

Airway structure and function in Eisenmenger's syndrome.

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McKay, K O; Johnson, P R; Black, J L; Glanville, A R; Armour, C L

1998-10-01

The responsiveness of airways from patients with Eisenmenger's syndrome (n = 5) was compared with that in airways from organ donors (n = 10). Enhanced contractile responses to cholinergic stimulation were found in airways from patients with Eisenmenger's syndrome. The maximal responses to acetylcholine, carbachol, and parasympathetic nerve stimulation in airway tissue from these patients were 221%, 139%, and 152%, respectively, of the maximal responses obtained in donor tissue. Further, relaxation responses to isoproterenol and levocromakalim were absent (n = 2) or markedly impaired (n = 3) in airways from patients with Eisenmenger's syndrome. This attenuated relaxation response was nonspecific in that it was also absent after vasoactive intestinal peptide, sodium nitroprusside, papaverine, and electrical field application. These observations can most likely be explained by a decrease in intrinsic smooth muscle tone, as precontraction of airways revealed relaxation responses that were equivalent to those obtained in donor tissues. Morphometric analysis of tissues used for the functional studies revealed no differences in the airway dimensions (internal perimeter) or airway wall components (e.g., smooth muscle, cartilage) or total area to explain these observations. Although the mechanism for this observed decrease in intrinsic airway smooth muscle tone is not certain, it may be due to alteration in the substructure of the airway wall or, alternatively, may result from the continued release of depressant factors in the vicinity of the smooth muscle which permanently alters smooth muscle responsiveness.

Eosinophils enhance WNT-5a and TGF-β1 genes expression in airway smooth muscle cells and promote their proliferation by increased extracellular matrix proteins production in asthma

NARCIS (Netherlands)

Januskevicius, Andrius; Vaitkiene, Simona; Gosens, Reinoud; Janulaityte, Ieva; Hoppenot, Deimante; Sakalauskas, Raimundas; Malakauskas, Kestutis

2016-01-01

BACKGROUND: Recent studies have suggested that eosinophils may have a direct effect on airway smooth muscle cells (ASMC), causing their proliferation in patients with asthma, but the precise mechanism of the interaction between these cells remains unknown. We propose that changes in Wnt signaling

Dense-body aggregates as plastic structures supporting tension in smooth muscle cells.

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Zhang, Jie; Herrera, Ana M; Paré, Peter D; Seow, Chun Y

2010-11-01

The wall of hollow organs of vertebrates is a unique structure able to generate active tension and maintain a nearly constant passive stiffness over a large volume range. These properties are predominantly attributable to the smooth muscle cells that line the organ wall. Although smooth muscle is known to possess plasticity (i.e., the ability to adapt to large changes in cell length through structural remodeling of contractile apparatus and cytoskeleton), the detailed structural basis for the plasticity is largely unknown. Dense bodies, one of the most prominent structures in smooth muscle cells, have been regarded as the anchoring sites for actin filaments, similar to the Z-disks in striated muscle. Here, we show that the dense bodies and intermediate filaments formed cable-like structures inside airway smooth muscle cells and were able to adjust the cable length according to cell length and tension. Stretching the muscle cell bundle in the relaxed state caused the cables to straighten, indicating that these intracellular structures were connected to the extracellular matrix and could support passive tension. These plastic structures may be responsible for the ability of smooth muscle to maintain a nearly constant tensile stiffness over a large length range. The finding suggests that the structural plasticity of hollow organs may originate from the dense-body cables within the smooth muscle cells.

Human Lung Mast Cell Products Regulate Airway Smooth Muscle CXCL10 Levels.

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Alkhouri, H; Cha, V; Tong, K; Moir, L M; Armour, C L; Hughes, J M

2014-01-01

In asthma, the airway smooth muscle (ASM) produces CXCL10 which may attract CXCR3(+) mast/T cells to it. Our aim was to investigate the effects of mast cell products on ASM cell CXCL10 production. ASM cells from people with and without asthma were stimulated with IL-1 β , TNF- α , and/or IFN γ and treated with histamine (1-100  μ M) ± chlorpheniramine (H1R antagonist; 1  μ M) or ranitidine (H2R antagonist; 50  μ M) or tryptase (1 nM) ± leupeptin (serine protease inhibitor; 50  μ M), heat-inactivated tryptase, or vehicle for 4 h or 24 h. Human lung mast cells (MC) were isolated and activated with IgE/anti-IgE and supernatants were collected after 2 h or 24 h. The supernatants were added to ASM cells for 48 h and ASM cell CXCL10 production detected using ELISA (protein) and real-time PCR (mRNA). Histamine reduced IL-1 β /TNF- α -induced CXCL10 protein, but not mRNA, levels independent of H1 and H2 receptor activation, whereas tryptase and MC 2 h supernatants reduced all cytokine-induced CXCL10. Tryptase also reduced CXCL10 levels in a cell-free system. Leupeptin inhibited the effects of tryptase and MC 2 h supernatants. MC 24 h supernatants contained TNF- α and amplified IFN γ -induced ASM cell CXCL10 production. This is the first evidence that MC can regulate ASM cell CXCL10 production and its degradation. Thus MC may regulate airway myositis in asthma.

Serum IgE Induced Airway Smooth Muscle Cell Remodeling Is Independent of Allergens and Is Prevented by Omalizumab

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Roth, Michael; Zhao, Feng; Zhong, Jun; Lardinois, Didier; Tamm, Michael

2015-01-01

Background Airway wall remodeling in allergic asthma is reduced after treatment with humanized anti-IgE-antibodies. We reported earlier that purified IgE, without the presence of allergens, is sufficient to induce airway wall remodeling due to airway smooth muscle cell (ASMC) activity deposing extracellular matrix. Objective We postulate that IgE contained in serum of allergic asthma patients, in the absence of allergens, stimulates ASMC remodeling activities and can be prevented by anti-IgE antibodies. Methods Isolated human ASMC were exposed to serum obtained from: (i) healthy controls, or patients with (ii) allergic asthma, (iii) non-allergic asthma, and (iv) atopic non-asthma patients. Proliferation and the deposition of collagens and fibronectin were determined after 3 and 5 days. Results Serum from patients with allergies significantly stimulated: (i) ASMC proliferation, (ii) deposition of collagen type-I (48 hours) and (iii) of fibronectin (24 hours). One hour pre-incubation with Omalizumab prevented these three effects of allergic serum, but had no significant effect on serum from healthy donors or non-allergic asthma patients. Interestingly, the addition of allergens did not further increase any of the IgE effects. Conclusion and Clinical Relevance Our data provides experimental evidence that the beneficial effect of Omalizumab on airway wall remodeling and improved lung function may be due to its direct action on IgE bound ASMC. PMID:26332463

Serum IgE Induced Airway Smooth Muscle Cell Remodeling Is Independent of Allergens and Is Prevented by Omalizumab.

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Michael Roth

Full Text Available Airway wall remodeling in allergic asthma is reduced after treatment with humanized anti-IgE-antibodies. We reported earlier that purified IgE, without the presence of allergens, is sufficient to induce airway wall remodeling due to airway smooth muscle cell (ASMC activity deposing extracellular matrix.We postulate that IgE contained in serum of allergic asthma patients, in the absence of allergens, stimulates ASMC remodeling activities and can be prevented by anti-IgE antibodies.Isolated human ASMC were exposed to serum obtained from: (i healthy controls, or patients with (ii allergic asthma, (iii non-allergic asthma, and (iv atopic non-asthma patients. Proliferation and the deposition of collagens and fibronectin were determined after 3 and 5 days.Serum from patients with allergies significantly stimulated: (i ASMC proliferation, (ii deposition of collagen type-I (48 hours and (iii of fibronectin (24 hours. One hour pre-incubation with Omalizumab prevented these three effects of allergic serum, but had no significant effect on serum from healthy donors or non-allergic asthma patients. Interestingly, the addition of allergens did not further increase any of the IgE effects.Our data provides experimental evidence that the beneficial effect of Omalizumab on airway wall remodeling and improved lung function may be due to its direct action on IgE bound ASMC.

PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells

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Halayko Andrew J

2009-09-01

Full Text Available Abstract Background Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8. IL-8 production is in part regulated via activation of Gq-and Gs-coupled receptors. Here we study the role of the cyclic AMP (cAMP effectors protein kinase A (PKA and exchange proteins directly activated by cAMP (Epac1 and Epac2 in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response. Methods IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases, U0126 (extracellular signal-regulated kinases ERK1/2 and Rp-8-CPT-cAMPS (PKA. The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used. Results The β2-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP

Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

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Matsuzaki, Shinichi [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Ishizuka, Tamotsu, E-mail: tamotsui@showa.gunma-u.ac.jp [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Yamada, Hidenori; Kamide, Yosuke; Hisada, Takeshi; Ichimonji, Isao; Aoki, Haruka; Yatomi, Masakiyo [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Komachi, Mayumi [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tsurumaki, Hiroaki; Ono, Akihiro; Koga, Yasuhiko [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Dobashi, Kunio [Gunma University Graduate School of Health Sciences, Maebashi 371-8511 (Japan); Mogi, Chihiro; Sato, Koichi; Tomura, Hideaki [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Mori, Masatomo [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan)

2011-10-07

Highlights: {yields} The involvement of extracellular acidification in airway remodeling was investigated. {yields} Extracellular acidification alone induced CTGF production in human ASMCs. {yields} Extracellular acidification enhanced TGF-{beta}-induced CTGF production in human ASMCs. {yields} Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. {yields} OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-{beta}-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G{sub q/11} protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP{sub 3}) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G{sub q/11} protein and inositol-1,4,5-trisphosphate-induced Ca{sup 2+} mobilization in human ASMCs.

Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

International Nuclear Information System (INIS)

Matsuzaki, Shinichi; Ishizuka, Tamotsu; Yamada, Hidenori; Kamide, Yosuke; Hisada, Takeshi; Ichimonji, Isao; Aoki, Haruka; Yatomi, Masakiyo; Komachi, Mayumi; Tsurumaki, Hiroaki; Ono, Akihiro; Koga, Yasuhiko; Dobashi, Kunio; Mogi, Chihiro; Sato, Koichi; Tomura, Hideaki; Mori, Masatomo; Okajima, Fumikazu

2011-01-01

Highlights: → The involvement of extracellular acidification in airway remodeling was investigated. → Extracellular acidification alone induced CTGF production in human ASMCs. → Extracellular acidification enhanced TGF-β-induced CTGF production in human ASMCs. → Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. → OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-β-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G q/11 protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP 3 ) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G q/11 protein and inositol-1,4,5-trisphosphate-induced Ca 2+ mobilization in human ASMCs.

Logarithmic superposition of force response with rapid length changes in relaxed porcine airway smooth muscle.

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Ijpma, G; Al-Jumaily, A M; Cairns, S P; Sieck, G C

2010-12-01

We present a systematic quantitative analysis of power-law force relaxation and investigate logarithmic superposition of force response in relaxed porcine airway smooth muscle (ASM) strips in vitro. The term logarithmic superposition describes linear superposition on a logarithmic scale, which is equivalent to multiplication on a linear scale. Additionally, we examine whether the dynamic response of contracted and relaxed muscles is dominated by cross-bridge cycling or passive dynamics. The study shows the following main findings. For relaxed ASM, the force response to length steps of varying amplitude (0.25-4% of reference length, both lengthening and shortening) are well-fitted with power-law functions over several decades of time (10⁻² to 10³ s), and the force response after consecutive length changes is more accurately fitted assuming logarithmic superposition rather than linear superposition. Furthermore, for sinusoidal length oscillations in contracted and relaxed muscles, increasing the oscillation amplitude induces greater hysteresivity and asymmetry of force-length relationships, whereas increasing the frequency dampens hysteresivity but increases asymmetry. We conclude that logarithmic superposition is an important feature of relaxed ASM, which may facilitate a more accurate prediction of force responses in the continuous dynamic environment of the respiratory system. In addition, the single power-function response to length changes shows that the dynamics of cross-bridge cycling can be ignored in relaxed muscle. The similarity in response between relaxed and contracted states implies that the investigated passive dynamics play an important role in both states and should be taken into account.

Endogenous laminin is required for human airway smooth muscle cell maturation

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Tran Thai

2006-09-01

Full Text Available Abstract Background Airway smooth muscle (ASM contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells. Methods Human ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured. Results Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of α2, β1 and γ1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype. Conclusion While earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the

Airway basement membrane perimeter in human airways is not a constant; potential implications for airway remodeling in asthma.

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McParland, Brent E; Paré, Peter D; Johnson, Peter R A; Armour, Carol L; Black, Judith L

2004-08-01

Many studies that demonstrate an increase in airway smooth muscle in asthmatic patients rely on the assumption that bronchial internal perimeter (P(i)) or basement membrane perimeter (P(bm)) is a constant, i.e., not affected by fixation pressure or the degree of smooth muscle shortening. Because it is the basement membrane that has been purported to be the indistensible structure, this study examines the assumption that P(bm) is not affected by fixation pressure. P(bm) was determined for the same human airway segment (n = 12) fixed at distending pressures of 0 cmH(2)O and 21 cmH(2)O in the absence of smooth muscle tone. P(bm) for the segment fixed at 0 cmH(2)O was determined morphometrically, and the P(bm) for the same segment, had the segment been fixed at 21 cmH(2)O, was predicted from knowing the luminal volume and length of the airway when distended to 21 cmH(2)O (organ bath-derived P(i)). To ensure an accurate transformation of the organ bath-derived P(i) value to a morphometry-derived P(bm) value, had the segment been fixed at 21 cmH(2)O, the relationship between organ bath-derived P(i) and morphometry-derived P(bm) was determined for five different bronchial segments distended to 21 cmH(2)O and fixed at 21 cmH(2)O (r(2) = 0.99, P < 0.0001). Mean P(bm) for bronchial segments fixed at 0 cmH(2)O was 9.4 +/- 0.4 mm, whereas mean predicted P(bm), had the segments been fixed at 21 cmH(2)O, was 14.1 +/- 0.5 mm (P < 0.0001). This indicates that P(bm) is not a constant when isolated airway segments without smooth muscle tone are fixed distended to 21 cmH(2)O. The implication of these results is that the increase in smooth muscle mass in asthma may have been overestimated in some previous studies. Therefore, further studies are required to examine the potential artifact using whole lungs with and without abolition of airway smooth muscle tone and/or inflation.

Estrogen receptor beta signaling inhibits PDGF induced human airway smooth muscle proliferation.

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Ambhore, Nilesh Sudhakar; Katragadda, Rathnavali; Raju Kalidhindi, Rama Satyanarayana; Thompson, Michael A; Pabelick, Christina M; Prakash, Y S; Sathish, Venkatachalem

2018-04-20

Airway smooth muscle (ASM) cell hyperplasia driven by persistent inflammation is a hallmark feature of remodeling in asthma. Sex steroid signaling in the lungs is of considerable interest, given epidemiological data showing more asthma in pre-menopausal women and aging men. Our previous studies demonstrated that estrogen receptor (ER) expression increases in asthmatic human ASM; however, very limited data are available regarding differential roles of ERα vs. ERβ isoforms in human ASM cell proliferation. In this study, we evaluated the effect of selective ERα and ERβ modulators on platelet-derived growth factor (PDGF)-stimulated ASM proliferation and the mechanisms involved. Asthmatic and non-asthmatic primary human ASM cells were treated with PDGF, 17β-estradiol, ERα-agonist and/or ERβ-agonist and/or G-protein-coupled estrogen receptor 30 (GPR30/GPER) agonist and proliferation was measured using MTT and CyQuant assays followed by cell cycle analysis. Transfection of small interfering RNA (siRNA) ERα and ERβ significantly altered the human ASM proliferation. The specificity of siRNA transfection was confirmed by Western blot analysis. Gene and protein expression of cell cycle-related antigens (PCNA and Ki67) and C/EBP were measured by RT-PCR and Western analysis, along with cell signaling proteins. PDGF significantly increased ASM proliferation in non-asthmatic and asthmatic cells. Treatment with PPT showed no significant effect on PDGF-induced proliferation, whereas WAY interestingly suppressed proliferation via inhibition of ERK1/2, Akt, and p38 signaling. PDGF-induced gene expression of PCNA, Ki67 and C/EBP in human ASM was significantly lower in cells pre-treated with WAY. Furthermore, WAY also inhibited PDGF-activated PCNA, C/EBP, cyclin-D1, and cyclin-E. Overall, we demonstrate ER isoform-specific signaling in the context of ASM proliferation. Activation of ERβ can diminish remodeling in human ASM by inhibiting pro-proliferative signaling pathways

Fibrocyte localization to the airway smooth muscle is a feature of asthma.

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Saunders, Ruth; Siddiqui, Salman; Kaur, Davinder; Doe, Camille; Sutcliffe, Amanda; Hollins, Fay; Bradding, Peter; Wardlaw, Andrew; Brightling, Christopher E

2009-02-01

Airway smooth muscle (ASM) hyperplasia is a hallmark of asthma that is associated with disease severity and persistent airflow obstruction. We sought to investigate whether fibrocytes, a population of peripheral blood mesenchymal progenitors, are recruited to the ASM compartment in asthma. We assessed the number of fibrocytes in bronchial biopsy specimens and peripheral blood from subjects with mild-to-severe refractory asthma versus healthy control subjects. In vitro we investigated potential mechanisms controlling fibrocyte migration toward the ASM bundle. Fifty-one subjects with asthma and 33 control subjects were studied. In bronchial biopsy specimens, the number of fibrocytes was increased in the lamina propria of subjects with severe refractory asthma (median [interquartile range] number, 1.9/mm(2) [1.7/mm(2)]) versus healthy control subjects (median [interquartile range] number, 0/mm(2) [0.3/mm(2)], P interquartile range] number, 3.8/mm(2) [9.4/mm(2)]; subjects with mild-to-moderate asthma, median [interquartile range] number, 1.1/mm(2) [2.4/mm(2)]); healthy control subjects, (median [interquartile range] number, 0/mm(2) [0/mm(2)]); P = .0004). In the peripheral blood the fibrocyte number was also increased in subjects with severe refractory asthma (median [interquartile range] number, 1.4 x 10(4)/mL [2.6 x 10(4)/mL]) versus healthy control subjects (median [interquartile range] number, 0.4 x 10(4)/mL [1.0 x 10(4)/mL], P = .002). We identified that in vitro ASM promotes fibrocyte chemotaxis and chemokinesis (distance of migration after 4.5 hours, 31 microm [2.9 microm] vs 17 microm [2.4 microm], P = .0001), which was in part mediated by platelet-derived growth factor (mean inhibition by neutralizing antibody, 16% [95% CI, 2% to 32%], P = .03) but not by activation of chemokine receptors. This study provides the first evidence that fibrocytes are present in the ASM compartment in asthma and that ASM can augment fibrocyte migration. The importance of

CD38/cADPR Signaling Pathway in Airway Disease: Regulatory Mechanisms

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Deepak A. Deshpande

2018-01-01

Full Text Available Asthma is an inflammatory disease in which proinflammatory cytokines have a role in inducing abnormalities of airway smooth muscle function and in the development of airway hyperresponsiveness. Inflammatory cytokines alter calcium (Ca2+ signaling and contractility of airway smooth muscle, which results in nonspecific airway hyperresponsiveness to agonists. In this context, Ca2+ regulatory mechanisms in airway smooth muscle and changes in these regulatory mechanisms encompass a major component of airway hyperresponsiveness. Although dynamic Ca2+ regulation is complex, phospholipase C/inositol tris-phosphate (PLC/IP3 and CD38-cyclic ADP-ribose (CD38/cADPR are two major pathways mediating agonist-induced Ca2+ regulation in airway smooth muscle. Altered CD38 expression or enhanced cyclic ADP-ribosyl cyclase activity associated with CD38 contributes to human pathologies such as asthma, neoplasia, and neuroimmune diseases. This review is focused on investigations on the role of CD38-cyclic ADP-ribose signaling in airway smooth muscle in the context of transcriptional and posttranscriptional regulation of CD38 expression. The specific roles of transcription factors NF-kB and AP-1 in the transcriptional regulation of CD38 expression and of miRNAs miR-140-3p and miR-708 in the posttranscriptional regulation and the underlying mechanisms of such regulation are discussed.

CD38/cADPR Signaling Pathway in Airway Disease: Regulatory Mechanisms

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Deshpande, Deepak A.; Guedes, Alonso G. P.; Graeff, Richard; Dogan, Soner; Subramanian, Subbaya; Walseth, Timothy F.

2018-01-01

Asthma is an inflammatory disease in which proinflammatory cytokines have a role in inducing abnormalities of airway smooth muscle function and in the development of airway hyperresponsiveness. Inflammatory cytokines alter calcium (Ca2+) signaling and contractility of airway smooth muscle, which results in nonspecific airway hyperresponsiveness to agonists. In this context, Ca2+ regulatory mechanisms in airway smooth muscle and changes in these regulatory mechanisms encompass a major component of airway hyperresponsiveness. Although dynamic Ca2+ regulation is complex, phospholipase C/inositol tris-phosphate (PLC/IP3) and CD38-cyclic ADP-ribose (CD38/cADPR) are two major pathways mediating agonist-induced Ca2+ regulation in airway smooth muscle. Altered CD38 expression or enhanced cyclic ADP-ribosyl cyclase activity associated with CD38 contributes to human pathologies such as asthma, neoplasia, and neuroimmune diseases. This review is focused on investigations on the role of CD38-cyclic ADP-ribose signaling in airway smooth muscle in the context of transcriptional and posttranscriptional regulation of CD38 expression. The specific roles of transcription factors NF-kB and AP-1 in the transcriptional regulation of CD38 expression and of miRNAs miR-140-3p and miR-708 in the posttranscriptional regulation and the underlying mechanisms of such regulation are discussed. PMID:29576747

GM-CSF production from human airway smooth muscle cells is potentiated by human serum

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Maria B. Sukkar

2000-01-01

Full Text Available Recent evidence suggests that airway smooth muscle cells (ASMC actively participate in the airway inflammatory process in asthma. Interleukin–1β (IL–1β and tumour necrosis factor–α (TNF–α induce ASMC to release inflammatory mediators in vitro. ASMC mediator release in vivo, however, may be influenced by features of the allergic asthmatic phenotype. We determined whether; (1 allergic asthmatic serum (AAS modulates ASMC mediator release in response to IL–1β and TNF–α, and (2 IL–1β/TNF–α prime ASMC to release mediators in response to AAS. IL–5 and GMCSF were quantified by ELISA in culture supernatants of; (1 ASMC pre-incubated with either AAS, non-allergic non-asthmatic serum (NAS or MonomedTM (a serum substitute and subsequently stimulated with IL–1β and TNF–α and (2 ASMC stimulated with IL–1β/TNF–α and subsequently exposed to either AAS, NAS or MonomedTM. IL-1g and TNF–α induced GM-CSF release in ASMC pre-incubated with AAS was not greater than that in ASMC pre-incubated with NAS or MonomedTM. IL–1β and TNF–α, however, primed ASMC to release GM-CSF in response to human serum. GM-CSF production following IL–1β/TNF–α and serum exposure (AAS or NAS was significantly greater than that following IL–1β /TNF–α and MonomedTM exposure or IL–1β/TNF–α exposure only. Whilst the potentiating effects of human serum were not specific to allergic asthma, these findings suggest that the secretory capacity of ASMC may be up-regulated during exacerbations of asthma, where there is evidence of vascular leakage.

Anti-smooth muscle antibody

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... gov/ency/article/003531.htm Anti-smooth muscle antibody To use the sharing features on this page, please enable JavaScript. Anti-smooth muscle antibody is a blood test that detects the presence ...

Opportunities to Target Specific Contractile Abnormalities with Smooth Muscle Protein Kinase Inhibitors

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Annegret Ulke-Lemée

2010-05-01

Full Text Available Smooth muscle is a major component of most hollow organ systems (e.g., airways, vasculature, bladder and gut/gastrointestine; therefore, the coordinated regulation of contraction is a key property of smooth muscle. When smooth muscle functions normally, it contributes to general health and wellness, but its dysfunction is associated with morbidity and mortality. Rho-associated protein kinase (ROCK is central to calcium-independent, actomyosin-mediated contractile force generation in the vasculature, thereby playing a role in smooth muscle contraction, cell motility and adhesion. Recent evidence supports an important role for ROCK in the increased vasoconstriction and remodeling observed in various models of hypertension. This review will provide a commentary on the development of specific ROCK inhibitors and their clinical application. Fasudil will be discussed as an example of bench-to-bedside development of a clinical therapeutic that is used to treat conditions of vascular hypercontractility. Due to the wide spectrum of biological processes regulated by ROCK, many additional clinical indications might also benefit from ROCK inhibition. Apart from the importance of ROCK in smooth muscle contraction, a variety of other protein kinases are known to play similar roles in regulating contractile force. The zipper-interacting protein kinase (ZIPK and integrin-linked kinase (ILK are two well-described regulators of contraction. The relative contribution of each kinase to contraction depends on the muscle bed as well as hormonal and neuronal stimulation. Unfortunately, specific inhibitors for ZIPK and ILK are still in the development phase, but the success of fasudil suggests that inhibitors for these other kinases may also have valuable clinical applications. Notably, the directed inhibition of ZIPK with a pseudosubstrate molecule shows unexpected effects on the contractility of gastrointestinal smooth muscle.

Phenotype and Functional Features of Human Telomerase Reverse Transcriptase Immortalized Human Airway Smooth Muscle Cells from Asthmatic and Non-Asthmatic Donors.

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Burgess, J K; Ketheson, A; Faiz, A; Limbert Rempel, K A; Oliver, B G; Ward, J P T; Halayko, A J

2018-01-16

Asthma is an obstructive respiratory disease characterised by chronic inflammation with airway hyperresponsiveness. In asthmatic airways, there is an increase in airway smooth muscle (ASM) cell bulk, which differs from non-asthmatic ASM in characteristics. This study aimed to assess the usefulness of hTERT immortalisation of human ASM cells as a research tool. Specifically we compared proliferative capacity, inflammatory mediator release and extracellular matrix (ECM) production in hTERT immortalised and parent primary ASM cells from asthmatic and non-asthmatic donors. Our studies revealed no significant differences in proliferation, IL-6 and eotaxin-1 production, or CTGF synthesis between donor-matched parent and hTERT immortalised ASM cell lines. However, deposition of ECM proteins fibronectin and fibulin-1 was significantly lower in immortalised ASM cells compared to corresponding primary cells. Notably, previously reported differences in proliferation and inflammatory mediator release between asthmatic and non-asthmatic ASM cells were retained, but excessive ECM protein deposition in asthmatic ASM cells was lost in hTERT ASM cells. This study shows that hTERT immortalised ASM cells mirror primary ASM cells in proliferation and inflammatory profile characteristics. Moreover, we demonstrate both strengths and weaknesses of this immortalised cell model as a representation of primary ASM cells for future asthma pathophysiological research.

Insulin induces airway smooth muscle contraction

NARCIS (Netherlands)

Schaafsma, D.; Gosens, R.; Ris, J. M.; Zaagsma, J.; Meurs, H.; Nelemans, S. A.

Background and purpose: Recently, the use of inhaled insulin formulations for the treatment of type I and type II diabetes has been approved in Europe and in the United States. For regular use, it is critical that airway function remains unimpaired in response to insulin exposure. Experimental

Impaired Bronchoprotection Is Not Induced by Increased Smooth Muscle Mass in Chronic Treatment In Vivo with Formoterol in Asthmatic Mouse Model

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W Luo

2014-09-01

Full Text Available Objective: Inhaling β2-adrenoceptor agonist is first-line asthma treatment, which is used for both acute relief and prevention of bronchoconstriction. However, chronic use of β-agonists results in impaired bronchoprotection and increasing occurrences of severe asthma exacerbation, even death in clinical practice. The mechanism of β-adrenoceptor hyposensitivity has not been thoroughly elucidated thus far. Bronchial smooth muscle contraction induces airway narrowing and also mediates airway inflammation. Moreover, bronchial smooth muscle mass significantly increases in asthmatics. We aimed to establish an asthmatic model that demonstrated that formoterol induced impaired bronchoprotection and to see whether increased smooth muscle mass played a role in it. Methods: We combined routine allergen challenging (seven weeks with repeated application of formoterol, formoterol plus budesonide or physiological saline in allergen-sensitized BALB/c mouse. The bronchoprotection mediated by β-agonist was measured in five consecutive weeks. Smooth muscle mass was shown by morphometric analysis, and α-actin expression was detected by western blot. Results: The trend of bronchoprotection was wavy in drug interventional groups, which initially increased and then decreased. Chronic treatment with formoterol significantly impaired bronchoprotection. According to the morphometric analysis and α-actin expression, no significant difference was detected in smooth muscle mass in all groups. Conclusion: This experiment successfully established that a chronic asthmatic mouse model, which manifested typical features of asthmatic patients, with chronic use of formoterol, results in a loss of bronchoprotection. No significant difference was detected in smooth muscle mass in all groups, which implied some subcellular signalling changes may be the key points.

Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle.

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West, Adrian R; Zaman, Nishat; Cole, Darren J; Walker, Matthew J; Legant, Wesley R; Boudou, Thomas; Chen, Christopher S; Favreau, John T; Gaudette, Glenn R; Cowley, Elizabeth A; Maksym, Geoffrey N

2013-01-01

Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.

The anti-proliferative and anti-inflammatory response of COPD airway smooth muscle cells to hydrogen sulfide.

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Perry, Mark M; Tildy, Bernadett; Papi, Alberto; Casolari, Paolo; Caramori, Gaetano; Rempel, Karen Limbert; Halayko, Andrew J; Adcock, Ian; Chung, Kian Fan

2018-05-09

COPD is a common, highly debilitating disease of the airways, primarily caused by smoking. Chronic inflammation and structural remodelling are key pathological features of this disease caused, in part, by the aberrant function of airway smooth muscle (ASM). We have previously demonstrated that hydrogen sulfide (H 2 S) can inhibit ASM cell proliferation and CXCL8 release, from cells isolated from non-smokers. We examined the effect of H 2 S upon ASM cells from COPD patients. ASM cells were isolated from non-smokers, smokers and patients with COPD (n = 9). Proliferation and cytokine release (IL-6 and CXCL8) of ASM was induced by FCS, and measured by bromodeoxyuridine incorporation and ELISA, respectively. Exposure of ASM to H 2 S donors inhibited FCS-induced proliferation and cytokine release, but was less effective upon COPD ASM cells compared to the non-smokers and smokers. The mRNA and protein expression of the enzymes responsible for endogenous H 2 S production (cystathionine-β-synthase [CBS] and 3-mercaptopyruvate sulphur transferase [MPST]) were inhibited by H 2 S donors. Finally, we report that exogenous H 2 S inhibited FCS-stimulated phosphorylation of ERK-1/2 and p38 mitogen activated protein kinases (MAPKs), in the non-smoker and smoker ASM cells, with little effect in COPD cells. H 2 S production provides a novel mechanism for the repression of ASM proliferation and cytokine release. The ability of COPD ASM cells to respond to H 2 S is attenuated in COPD ASM cells despite the presence of the enzymes responsible for H 2 S production.

Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

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Zhang, Rong; Jack, Gregory S; Rao, Nagesh; Zuk, Patricia; Ignarro, Louis J; Wu, Benjamin; Rodríguez, Larissa V

2012-03-01

Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion. Copyright © 2011 AlphaMed Press.

Calcium dynamics in vascular smooth muscle

OpenAIRE

Amberg, Gregory C.; Navedo, Manuel F.

2013-01-01

Smooth muscle cells are ultimately responsible for determining vascular luminal diameter and blood flow. Dynamic changes in intracellular calcium are a critical mechanism regulating vascular smooth muscle contractility. Processes influencing intracellular calcium are therefore important regulators of vascular function with physiological and pathophysiological consequences. In this review we discuss the major dynamic calcium signals identified and characterized in vascular smooth muscle cells....

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening.

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An, Steven S; Askovich, Peter S; Zarembinski, Thomas I; Ahn, Kwangmi; Peltier, John M; von Rechenberg, Moritz; Sahasrabudhe, Sudhir; Fredberg, Jeffrey J

2011-01-13

A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma. Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds. Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo. This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

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von Rechenberg Moritz

2011-01-01

Full Text Available Abstract Background A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20 and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma. Methods Using a high-throughput fluorescence polarization (FP assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM. Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds. Results Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo. Conclusions This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

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Zaagsma Johan

2005-07-01

Full Text Available Abstract Background In addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction. Methods Growth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK and cyclooxygenase (COX to these reponses was established, using the inhibitors Y-27632 (1 μM, U-0126 (3 μM and indomethacin (3 μM, respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F2α (PGF2α and prostaglandin E2 (PGE2 was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 μM and the selective EP1-antagonist AH-6809 (10 μM on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF2α-and PGE2-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay. Results Epidermal growth factor (EGF-and platelet-derived growth factor (PDGF-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF2α-and PGE2-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF2α-and PGE2-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 μM significantly

Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation

DEFF Research Database (Denmark)

Nassini, Romina; Pedretti, Pamela; Moretto, Nadia

2012-01-01

The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic...... inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1.By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express...... functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells...

Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca(2+) regulation in airway smooth muscle (ASM).

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Delmotte, Philippe; Sieck, Gary C

2015-02-01

Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca(2+) ([Ca(2+)]cyt) responses to agonist stimulation and Ca(2+) sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca(2+)]cyt induced by agonists leads to a transient increase in mitochondrial Ca(2+) ([Ca(2+)]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca(2+)]mito is blunted despite enhanced [Ca(2+)]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion-ER/SR coupling, decreased mitochondrial Ca(2+) buffering, mitochondrial fragmentation, and increased cell proliferation.

Link between vitamin D and airway remodeling

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Berraies A

2014-04-01

Full Text Available Anissa Berraies, Kamel Hamzaoui, Agnes HamzaouiPediatric Respiratory Diseases Department, Abderrahmen Mami Hospital, Ariana, and Research Unit 12SP15 Tunis El Manar University, Tunis, TunisiaAbstract: In the last decade, many epidemiologic studies have investigated the link between vitamin D deficiency and asthma. Most studies have shown that vitamin D deficiency increases the risk of asthma and allergies. Low levels of vitamin D have been associated with asthma severity and loss of control, together with recurrent exacerbations. Remodeling is an early event in asthma described as a consequence of production of mediators and growth factors by inflammatory and resident bronchial cells. Consequently, lung function is altered, with a decrease in forced expiratory volume in one second and exacerbated airway hyperresponsiveness. Subepithelial fibrosis and airway smooth muscle cell hypertrophy are typical features of structural changes in the airways. In animal models, vitamin D deficiency enhances inflammation and bronchial anomalies. In severe asthma of childhood, major remodeling is observed in patients with low vitamin D levels. Conversely, the antifibrotic and antiproliferative effects of vitamin D in smooth muscle cells have been described in several experiments. In this review, we briefly summarize the current knowledge regarding the relationship between vitamin D and asthma, and focus on its effect on airway remodeling and its potential therapeutic impact for asthma.Keywords: vitamin D, asthma, airway remodeling, airway smooth muscle, supplementation

Airway contractility and remodeling : Links to asthma symptoms

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West, Adrian R.; Syyong, Harley T.; Siddiqui, Sana; Pascoe, Chris D.; Murphy, Thomas M.; Maarsingh, Harm; Deng, Linhong; Maksym, Geoffrey N.; Bosse, Ynuk

Respiratory symptoms are largely caused by obstruction of the airways. In asthma, airway narrowing mediated by airway smooth muscle (ASM) contraction contributes significantly to obstruction. The spasmogens produced following exposure to environmental triggers, such as viruses or allergens, are

Airway remodeling and its reversibility in equine asthma

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Jean-Pierre Lavoie

2017-06-01

Full Text Available Despite effective therapies for controlling its clinical manifestations, human asthma remains an incurable disease. It is now recognized that inflammation induced structural changes (remodeling of the airways are responsible for the progressive loss of lung function in asthmatic patients. However, the peripheral airways, where most of the remodeling occurs in severe asthmatic patients, cannot be safely sampled in humans, and therefore, little is known of the effects of current therapies at reversing the established asthmatic remodeling, especially those occurring in the peripheral airways. Animal models have been studied to unravel etiological, immunopathological, and genetic attributes leading to asthma. However, experiments in which the disease is artificially induced have been shown to have limited translational potential for humans. To the contrary, horses naturally suffer from an asthma-like condition which shares marked similarities with human asthma making this model unique to investigate the kinetics, reversibility, as well as the physiological consequences of tissue remodeling (Bullone and Lavoie 2015. We reported an increased deposition of smooth muscle, collagen and elastic fibers in the peripheral airways of affected horses, which was correlated with the lung function (Herszberg et al., 2006; Setlakwe et al., 2014. The airway subepithelial collagen depositions were almost completely reversed with 6 to 12 months of treatment with either antigen avoidance or inhaled corticosteroids (ICS administration, and there was a modest (30% on average decrease in airway smooth muscle (Leclere et al., 2011. A recent study also found that ICS combined with long-acting ß2-agonists drugs (LABA and ICS monotherapy similarly induced a 30% decrease of the airway smooth muscle mass at 3 months (Buollone, 2017. However, only ICS/LABA and antigen avoidance decreased airway luminal neutrophilia. The findings indicate the enhance therapeutic effect of ICS

Nodular smooth muscle metaplasia in multiple peritoneal endometriosis

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Kim, Hyun-Soo; Yoon, Gun; Ha, Sang Yun; Song, Sang Yong

2015-01-01

We report here an unusual presentation of peritoneal endometriosis with smooth muscle metaplasia as multiple protruding masses on the lateral pelvic wall. Smooth muscle metaplasia is a common finding in rectovaginal endometriosis, whereas in peritoneal endometriosis, smooth muscle metaplasia is uncommon and its nodular presentation on the pelvic wall is even rarer. To the best of our knowledge, this is the first case of nodular smooth muscle metaplasia occurring in peritoneal endometriosis. A...

Effect of airway acidosis and alkalosis on airway vascular smooth muscle responsiveness to albuterol.

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Cancado, Jose E; Mendes, Eliana S; Arana, Johana; Horvath, Gabor; Monzon, Maria E; Salathe, Matthias; Wanner, Adam

2015-04-02

In vitro and animal experiments have shown that the transport and signaling of β2-adrenergic agonists are pH-sensitive. Inhaled albuterol, a hydrophilic β2-adrenergic agonist, is widely used for the treatment of obstructive airway diseases. Acute exacerbations of obstructive airway diseases can be associated with changes in ventilation leading to either respiratory acidosis or alkalosis thereby affecting albuterol responsiveness in the airway. The purpose of this study was to determine if airway pH has an effect on albuterol-induced vasodilation in the airway. Ten healthy volunteers performed the following respiratory maneuvers: quiet breathing, hypocapnic hyperventilation, hypercapnic hyperventilation, and eucapnic hyperventilation (to dissociate the effect of pH from the effect of ventilation). During these breathing maneuvers, exhaled breath condensate (EBC) pH and airway blood flow response to inhaled albuterol (ΔQ̇aw) were assessed. Mean ± SE EBC pH (units) and ΔQ̇aw (μl.min(-1).mL(-1)) were 6.4 ± 0.1 and 16.8 ± 1.9 during quiet breathing, 6.3 ± 0.1 and 14.5 ± 2.4 during eucapnic hyperventilation, 6.6 ± 0.2 and -0.2 ± 1.8 during hypocapnic hyperventilation (p = 0.02 and <0.01 vs. quiet breathing), and 5.9 ± 0.1 and 2.0 ± 1.5 during hypercapnic hyperventilation (p = 0.02 and <0.02 vs quiet breathing). Albuterol responsiveness in the airway as assessed by ΔQ̇aw is pH sensitive. The breathing maneuver associated with decreased and increased EBC pH both resulted in a decreased responsiveness independent of the level of ventilation. These findings suggest an attenuated response to hydrophilic β2-adrenergic agonists during airway disease exacerbations associated with changes in pH. Registered at clinicaltrials.gov: NCT01216748 .

Expression and activation of the oxytocin receptor in airway smooth muscle cells: Regulation by TNFα and IL-13

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Siddiqui Salman

2010-07-01

Full Text Available Abstract Background During pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs function in the presence and absence of IL-13 and TNFα, cytokines known to be important in asthma. Method Expression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL samples from healthy subjects and those with asthma. Results PCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL-13 and tumor necrosis factor (TNFα stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFα had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL fluid derived from healthy subjects as well as from those with asthma. Conclusion Taken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a

Functional phenotype of airway myocytes from asthmatic airways

NARCIS (Netherlands)

Wright, David B.; Trian, Thomas; Siddiqui, Sana; Pascoe, Chris D.; Ojo, Oluwaseun O.; Johnson, Jill R.; Dekkers, Bart G. J.; Dakshinamurti, Shyamala; Bagchi, Rushita; Burgess, Janette K.; Kanabar, Varsha

In asthma, the airway smooth muscle (ASM) cell plays a central role in disease pathogenesis through cellular changes which may impact on its microenvironment and alter ASM response and function. The answer to the long debated question of what makes a 'healthy' ASM cell become 'asthmatic' still

Nodular smooth muscle metaplasia in multiple peritoneal endometriosis.

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Kim, Hyun-Soo; Yoon, Gun; Ha, Sang Yun; Song, Sang Yong

2015-01-01

We report here an unusual presentation of peritoneal endometriosis with smooth muscle metaplasia as multiple protruding masses on the lateral pelvic wall. Smooth muscle metaplasia is a common finding in rectovaginal endometriosis, whereas in peritoneal endometriosis, smooth muscle metaplasia is uncommon and its nodular presentation on the pelvic wall is even rarer. To the best of our knowledge, this is the first case of nodular smooth muscle metaplasia occurring in peritoneal endometriosis. As observed in this case, when performing laparoscopic surgery in order to excise malignant tumors of intra-abdominal or pelvic organs, it can be difficult for surgeons to distinguish the metastatic tumors from benign nodular pelvic wall lesions, including endometriosis, based on the gross findings only. Therefore, an intraoperative frozen section biopsy of the pelvic wall nodules should be performed to evaluate the peritoneal involvement by malignant tumors. Moreover, this report implies that peritoneal endometriosis, as well as rectovaginal endometriosis, can clinically present as nodular lesions if obvious smooth muscle metaplasia is present. The pathological investigation of smooth muscle cells in peritoneal lesions can contribute not only to the precise diagnosis but also to the structure and function of smooth muscle cells and related cells involved in the histogenesis of peritoneal endometriosis.

Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement

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Alrashdan, Yazan A.; Alkhouri, Hatem; Chen, Emily; Lalor, Daniel J.; Poniris, Maree; Henness, Sheridan; Brightling, Christopher E.; Burgess, Janette K.; Armour, Carol L.; Ammit, Alaina J.

2012-01-01

CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma. PMID:22387292

Role of Smooth Muscle in Intestinal Inflammation

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Stephen M Collins

1996-01-01

Full Text Available The notion that smooth muscle function is altered in inflammation is prompted by clinical observations of altered motility in patients with inflammatory bowel disease (IBD. While altered motility may reflect inflammation-induced changes in intrinsic or extrinsic nerves to the gut, changes in gut hormone release and changes in muscle function, recent studies have provided in vitro evidence of altered muscle contractility in muscle resected from patients with ulcerative colitis or Crohn’s disease. In addition, the observation that smooth muscle cells are more numerous and prominent in the strictured bowel of IBD patients compared with controls suggests that inflammation may alter the growth of intestinal smooth muscle. Thus, inflammation is associated with changes in smooth muscle growth and contractility that, in turn, contribute to important symptoms of IBD including diarrhea (from altered motility and pain (via either altered motility or stricture formation. The involvement of smooth muscle in this context may be as an innocent bystander, where cells and products of the inflammatory process induce alterations in muscle contractility and growth. However, it is likely that intestinal muscle cells play a more active role in the inflammatory process via the elaboration of mediators and trophic factors, including cytokines, and via the production of collagen. The concept of muscle cells as active participants in the intestinal inflammatory process is a new concept that is under intense study. This report summarizes current knowledge as it relates to these two aspects of altered muscle function (growth and contractility in the inflamed intestine, and will focus on mechanisms underlying these changes, based on data obtained from animal models of intestinal inflammation.

The role of inflammation resolution speed in airway smooth muscle mass accumulation in asthma: insight from a theoretical model.

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Igor L Chernyavsky

Full Text Available Despite a large amount of in vitro data, the dynamics of airway smooth muscle (ASM mass increase in the airways of patients with asthma is not well understood. Here, we present a novel mathematical model that describes qualitatively the growth dynamics of ASM cells over short and long terms in the normal and inflammatory environments typically observed in asthma. The degree of ASM accumulation can be explained by an increase in the rate at which ASM cells switch between non-proliferative and proliferative states, driven by episodic inflammatory events. Our model explores the idea that remodelling due to ASM hyperplasia increases with the frequency and magnitude of these inflammatory events, relative to certain sensitivity thresholds. It highlights the importance of inflammation resolution speed by showing that when resolution is slow, even a series of small exacerbation events can result in significant remodelling, which persists after the inflammatory episodes. In addition, we demonstrate how the uncertainty in long-term outcome may be quantified and used to design an optimal low-risk individual anti-proliferative treatment strategy. The model shows that the rate of clearance of ASM proliferation and recruitment factors after an acute inflammatory event is a potentially important, and hitherto unrecognised, target for anti-remodelling therapy in asthma. It also suggests new ways of quantifying inflammation severity that could improve prediction of the extent of ASM accumulation. This ASM growth model should prove useful for designing new experiments or as a building block of more detailed multi-cellular tissue-level models.

Aerobic metabolism on muscle contraction in porcine gastric smooth muscle.

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Kanda, Hidenori; Kaneda, Takeharu; Nagai, Yuta; Urakawa, Norimoto; Shimizu, Kazumasa

2018-05-18

Exposure to chronic hypoxic conditions causes various gastric diseases, including gastric ulcers. It has been suggested that gastric smooth muscle contraction is associated with aerobic metabolism. However, there are no reports on the association between gastric smooth muscle contraction and aerobic metabolism, and we have investigated this association in the present study. High K + - and carbachol (CCh)-induced muscle contractions involved increasing O 2 consumption. Aeration with N 2 (hypoxia) and NaCN significantly decreased high K + - and CCh-induced muscle contraction and O 2 consumption. In addition, hypoxia and NaCN significantly decreased creatine phosphate (PCr) contents in the presence of high K + . Moreover, decrease in CCh-induced contraction and O 2 consumption was greater than that of high K + . Our results suggest that hypoxia and NaCN inhibit high K + - and CCh-induced contractions in gastric fundus smooth muscles by decreasing O 2 consumption and intracellular PCr content. However, the inhibition of CCh-induced muscle contraction was greater than that of high K + -induced muscle contraction.

PM2.5 promotes human bronchial smooth muscle cell migration via the sonic hedgehog signaling pathway.

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Ye, Xiuqin; Hong, Wei; Hao, Binwei; Peng, Gongyong; Huang, Lingmei; Zhao, Zhuxiang; Zhou, Yumin; Zheng, Mengning; Li, Chenglong; Liang, Chunxiao; Yi, Erkang; Pu, Jinding; Li, Bing; Ran, Pixin

2018-03-02

The contribution of airway remodeling in chronic obstructive pulmonary disease (COPD) has been well documented, with airway smooth muscle cell proliferation and migration playing a role in the remodeling process. Here, we aimed to verify the effects of fine particulate matter (PM2.5) on human bronchial smooth muscle cell (HBSMC) migration and to explore the underlying signaling pathways. HBSMC apoptosis, proliferation and migration were measured using flow cytometry, cell counting and transwell migration assays, respectively. The role of the hedgehog pathway in cell migration was assessed by western blotting to measure the expression of Sonic hedgehog (Shh), Gli1 and Snail. Furthermore, siRNA was used to knock down Gli1 or Snail expression. PM2.5 induced HBSMC apoptosis in a dose-dependent manner, although certain concentrations of PM2.5 did not induce HBSMC proliferation or apoptosis. Interestingly, cell migration was stimulated by PM2.5 doses far below those that induced apoptosis. Additional experiments revealed that these PM2.5 doses enhanced the expression of Shh, Gli1 and Snail in HBSMCs. Furthermore, PM2.5-induced cell migration and protein expression were enhanced by recombinant Shh and attenuated by cyclopamine. Similar results were obtained by knocking down Gli1 or Snail. These findings suggest that PM2.5, which may exert its effects through the Shh signaling pathway, is necessary for the migration of HBSMCs. These data define a novel role for PM2.5 in airway remodeling in COPD.

Defective Resensitization in Human Airway Smooth Muscle Cells Evokes β-Adrenergic Receptor Dysfunction in Severe Asthma.

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Manveen K Gupta

Full Text Available β2-adrenergic receptor (β2AR agonists (β2-agonist are the most commonly used therapy for acute relief in asthma, but chronic use of these bronchodilators paradoxically exacerbates airway hyper-responsiveness. Activation of βARs by β-agonist leads to desensitization (inactivation by phosphorylation through G-protein coupled receptor kinases (GRKs which mediate β-arrestin binding and βAR internalization. Resensitization occurs by dephosphorylation of the endosomal βARs which recycle back to the plasma membrane as agonist-ready receptors. To determine whether the loss in β-agonist response in asthma is due to altered βAR desensitization and/or resensitization, we used primary human airway smooth muscle cells (HASMCs isolated from the lungs of non-asthmatic and fatal-asthmatic subjects. Asthmatic HASMCs have diminished adenylyl cyclase activity and cAMP response to β-agonist as compared to non-asthmatic HASMCs. Confocal microscopy showed significant accumulation of phosphorylated β2ARs in asthmatic HASMCs. Systematic analysis of desensitization components including GRKs and β-arrestin showed no appreciable differences between asthmatic and non-asthmatic HASMCs. However, asthmatic HASMC showed significant increase in PI3Kγ activity and was associated with reduction in PP2A activity. Since reduction in PP2A activity could alter receptor resensitization, endosomal fractions were isolated to assess the agonist ready β2ARs as a measure of resensitization. Despite significant accumulation of β2ARs in the endosomes of asthmatic HASMCs, endosomal β2ARs cannot robustly activate adenylyl cyclase. Furthermore, endosomes from asthmatic HASMCs are associated with significant increase in PI3Kγ and reduced PP2A activity that inhibits β2AR resensitization. Our study shows that resensitization, a process considered to be a homeostasis maintaining passive process is inhibited in asthmatic HASMCs contributing to β2AR dysfunction which may underlie

Antithetic regulation by β-adrenergic receptors of Gq receptor signaling via phospholipase C underlies the airway β-agonist paradox

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McGraw, Dennis W.; Almoosa, Khalid F.; Paul, Richard J.; Kobilka, Brian K.; Liggett, Stephen B.

2003-01-01

β-adrenergic receptors (βARs) relax airway smooth muscle and bronchodilate, but chronic β-agonist treatment in asthma causes increased sensitivity to airway constriction (hyperreactivity) and is associated with exacerbations. This paradox was explored using mice with ablated βAR genes (βAR–/–) and transgenic mice overexpressing airway smooth muscle β2AR (β2AR-OE) representing two extremes: absence and persistent activity of airway βAR. Unexpectedly, βAR–/– mice, lacking these bronchodilating ...

Thiazolidinediones inhibit airway smooth muscle release of the chemokine CXCL10: in vitro comparison with current asthma therapies

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Seidel Petra

2012-10-01

Full Text Available Abstract Background Activated mast cells are present within airway smooth muscle (ASM bundles in eosinophilic asthma. ASM production of the chemokine CXCL10 plays a role in their recruitment. Thus the effects of glucocorticoids (fluticasone, budesonide, long-acting β2-agonists (salmeterol, formoterol and thiazolidinediones (ciglitazone, rosiglitazone on CXCL10 production by ASM cells (ASMC from people with and without asthma were investigated in vitro. Methods Confluent serum-deprived cells were treated with the agents before and during cytokine stimulation for 0-24 h. CXCL10 protein/mRNA, IκB-α levels and p65 activity were measured using ELISA, RT PCR, immunoblotting and p65 activity assays respectively. Data were analysed using ANOVA followed by Fisher’s post-hoc test. Results Fluticasone and/or salmeterol at 1 and 100 nM inhibited CXCL10 release induced by IL-1β and TNF-α, but not IFNγ or all three cytokines (cytomix. The latter was also not affected by budesonide and formoterol. In asthmatic ASMC low salmeterol, but not formoterol, concentrations increased cytomix-induced CXCL10 release and at 0.01 nM enhanced NF-κB activity. Salmeterol 0.1nM together with fluticasone 0.1 and 10 nM still increased CXCL10 release. The thiazolidinediones ciglitazone and rosiglitazone (at 25 and 100 μM inhibited cytomix-induced CXCL10 release but these inhibitory effects were not prevented by the PPAR-g antagonist GW9662. Ciglitazone did not affect early NF-κB activity and CXCL10 mRNA production. Conclusions Thus the thiazolidinediones inhibited asthmatic ASMC CXCL10 release under conditions when common asthma therapies were ineffective or enhanced it. They may provide an alternative strategy to reduce mast cell-ASM interactions and restore normal airway physiology in asthma.

Thiazolidinediones inhibit airway smooth muscle release of the chemokine CXCL10: in vitro comparison with current asthma therapies.

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Seidel, Petra; Alkhouri, Hatem; Lalor, Daniel J; Burgess, Janette K; Armour, Carol L; Hughes, J Margaret

2012-10-04

Activated mast cells are present within airway smooth muscle (ASM) bundles in eosinophilic asthma. ASM production of the chemokine CXCL10 plays a role in their recruitment. Thus the effects of glucocorticoids (fluticasone, budesonide), long-acting β2-agonists (salmeterol, formoterol) and thiazolidinediones (ciglitazone, rosiglitazone) on CXCL10 production by ASM cells (ASMC) from people with and without asthma were investigated in vitro. Confluent serum-deprived cells were treated with the agents before and during cytokine stimulation for 0-24 h. CXCL10 protein/mRNA, IκB-α levels and p65 activity were measured using ELISA, RT PCR, immunoblotting and p65 activity assays respectively. Data were analysed using ANOVA followed by Fisher's post-hoc test. Fluticasone and/or salmeterol at 1 and 100 nM inhibited CXCL10 release induced by IL-1β and TNF-α, but not IFNγ or all three cytokines (cytomix). The latter was also not affected by budesonide and formoterol. In asthmatic ASMC low salmeterol, but not formoterol, concentrations increased cytomix-induced CXCL10 release and at 0.01 nM enhanced NF-κB activity. Salmeterol 0.1 nM together with fluticasone 0.1 and 10 nM still increased CXCL10 release. The thiazolidinediones ciglitazone and rosiglitazone (at 25 and 100 μM) inhibited cytomix-induced CXCL10 release but these inhibitory effects were not prevented by the PPAR-g antagonist GW9662. Ciglitazone did not affect early NF-κB activity and CXCL10 mRNA production. Thus the thiazolidinediones inhibited asthmatic ASMC CXCL10 release under conditions when common asthma therapies were ineffective or enhanced it. They may provide an alternative strategy to reduce mast cell-ASM interactions and restore normal airway physiology in asthma.

Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels.

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Gao, Wei; Cai, Liting; Xu, Xudong; Fan, Juxiang; Xue, Xiulei; Yan, Xuejiao; Qu, Qinrong; Wang, Xihua; Zhang, Chen; Wu, Guoqiu

2014-01-01

Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

Intracellular interactions of umeclidinium and vilanterol in human airway smooth muscle

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Shaikh N

2017-06-01

Full Text Available Nooreen Shaikh,1,2 Malcolm Johnson,3 David A Hall,4 Kian Fan Chung,1,2 John H Riley,3 Sally Worsley,5 Pankaj K Bhavsar1,2 1Experimental Studies, National Heart and Lung Institute, Imperial College London, 2Biomedical Research Unit, Royal Brompton and Harefield NHS Trust, London, 3Respiratory Global Franchise, GlaxoSmithKline, Uxbridge, 4Fibrosis and Lung Injury Development Planning Unit, GlaxoSmithKline, Stevenage, 5Respiratory Research & Development, GlaxoSmithKline, Uxbridge, UK Background: Intracellular mechanisms of action of umeclidinium (UMEC, a long-acting muscarinic receptor antagonist, and vilanterol (VI, a long-acting β2-adrenoceptor (β2R agonist, were investigated in target cells: human airway smooth-muscle cells (ASMCs. Materials and methods: ASMCs from tracheas of healthy lung-transplant donors were treated with VI, UMEC, UMEC and VI combined, or control compounds (salmeterol, propranolol, ICI 118.551, or methacholine [MCh]. Cyclic adenosine monophosphate (cAMP was measured using an enzyme-linked immunosorbent assay, intracellular free calcium ([Ca2+]i using a fluorescence assay, and regulator of G-protein signaling 2 (RGS2 messenger RNA using real-time quantitative polymerase chain reaction. Results: VI and salmeterol (10–12–10–6 M induced cAMP production from ASMCs in a concentration-dependent manner, which was greater for VI at all concentrations. β2R antagonism by propranolol or ICI 118.551 (10–12–10–4 M resulted in concentration-dependent inhibition of VI-induced cAMP production, and ICI 118.551 was more potent. MCh (5×10–6 M, 30 minutes attenuated VI-induced cAMP production (P<0.05, whereas pretreatment with UMEC (10–8 M, 1 hour restored the magnitude of VI-induced cAMP production. ASMC stimulation with MCh (10–11–5×10–6 M resulted in a concentration-dependent increase in [Ca2+]i, which was attenuated with UMEC pretreatment. Reduction of MCh-induced [Ca2+]i release was greater with UMEC + VI versus

trans-Caryophyllene, a Natural Sesquiterpene, Causes Tracheal Smooth Muscle Relaxation through Blockade of Voltage-Dependent Ca2+ Channels

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Jader Santos Cruz

2012-10-01

Full Text Available trans-Caryophyllene is a major component in the essential oils of various species of medicinal plants used in popular medicine in Brazil. It belongs to the chemical class of the sesquiterpenes and has been the subject of a number of studies. Here, we evaluated the effects of this compound in airway smooth muscle. The biological activities of trans-caryophyllene were examined in isolated bath organs to investigate the effect in basal tonus. Electromechanical and pharmacomechanical couplings were evaluated through the responses to K+ depolarization and exposure to acetylcholine (ACh, respectively. Isolated cells of rat tracheal smooth muscle were used to investigate trans-caryophyllene effects on voltage-dependent Ca2+ channels by using the whole-cell voltage-clamp configuration of the patch-clamp technique. trans-Caryophyllene showed more efficiency in the blockade of electromechanical excitation-contraction coupling while it has only minor inhibitory effect on pharmacomechanical coupling. Epithelium removal does not modify tracheal smooth muscle response elicited by trans-caryophyllene in the pharmacomechanical coupling. Under Ca2+-free conditions, pre-exposure to trans-caryophyllene did not reduce the contraction induced by ACh in isolated rat tracheal smooth muscle, regardless of the presence of intact epithelium. In the whole-cell configuration, trans-caryophyllene (3 mM, inhibited the inward Ba2+ current (IBa to approximately 50% of control levels. Altogether, our results demonstrate that trans-caryophyllene has anti-spasmodic activity on rat tracheal smooth muscle which could be explained, at least in part, by the voltage-dependent Ca2+ channels blockade.

Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone

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Walseth Timothy F

2008-03-01

Full Text Available Abstract Background CD38 is expressed in human airway smooth muscle (HASM cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-α. CD38 has a role in airway hyperresponsiveness, a hallmark of asthma, since deficient mice develop attenuated airway hyperresponsiveness compared to wild-type mice following intranasal challenges with cytokines such as IL-13 and TNF-α. Regulation of CD38 expression in HASM cells involves the transcription factor NF-κB, and glucocorticoids inhibit this expression through NF-κB-dependent and -independent mechanisms. In this study, we determined whether the transcriptional regulation of CD38 expression in HASM cells involves response elements within the promoter region of this gene. Methods We cloned a putative 3 kb promoter fragment of the human cd38 gene into pGL3 basic vector in front of a luciferase reporter gene. Sequence analysis of the putative cd38 promoter region revealed one NF-κB and several AP-1 and glucocorticoid response element (GRE motifs. HASM cells were transfected with the 3 kb promoter, a 1.8 kb truncated promoter that lacks the NF-κB and some of the AP-1 sites, or the promoter with mutations of the NF-κB and/or AP-1 sites. Using the electrophoretic mobility shift assays, we determined the binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB, AP-1, and GRE sites, and the specificity of this binding was confirmed by gel supershift analysis with appropriate antibodies. Results TNF-α induced a two-fold activation of the 3 kb promoter following its transfection into HASM cells. In cells transfected with the 1.8 kb promoter or promoter constructs lacking NF-κB and/or AP-1 sites or in the presence of dexamethasone, there was no induction in the presence of TNF-α. The binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB site and some of the six AP-1 sites was increased by TNF-α, and to

Evidence that CFTR is expressed in rat tracheal smooth muscle cells and contributes to bronchodilation

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Mettey Yvette

2006-08-01

Full Text Available Abstract Background The airway functions are profoundly affected in many diseases including asthma, chronic obstructive pulmonary disease (COPD and cystic fibrosis (CF. CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR gene, which normally encodes a multifunctional and integral membrane protein, the CF transmembrane conductance regulator (CFTR expressed in airway epithelial cells. Methods To demonstrate that CFTR is also expressed in tracheal smooth muscle cells (TSMC, we used iodide efflux assay to analyse the chloride transports in organ culture of rat TSMC, immunofluorescence study to localize CFTR proteins and isometric contraction measurement on isolated tracheal rings to observe the implication of CFTR in the bronchodilation. Results We characterized three different pathways stimulated by the cAMP agonist forskolin and the isoflavone agent genistein, by the calcium ionophore A23187 and by hypo-osmotic challenge. The pharmacology of the cAMP-dependent iodide efflux was investigated in detail. We demonstrated in rat TSMC that it is remarkably similar to that of the epithelial CFTR, both for activation (using three benzo [c]quinolizinium derivatives and for inhibition (glibenclamide, DPC and CFTRinh-172. Using rat tracheal rings, we observed that the activation of CFTR by benzoquinolizinium derivatives in TSMC leads to CFTRinh-172-sensitive bronchodilation after constriction with carbachol. An immunolocalisation study confirmed expression of CFTR in tracheal myocytes. Conclusion Altogether, these observations revealed that CFTR in the airways of rat is expressed not only in the epithelial cells but also in tracheal smooth muscle cells leading to the hypothesis that this ionic channel could contribute to bronchodilation.

Overexpression of soluble ADAM33 promotes a hypercontractile phenotype of the airway smooth muscle cell in rat

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Duan, Yiyuan; Long, Jiaoyue; Chen, Jun; Jiang, Xuemei; Zhu, Jian; Jin, Yang; Lin, Feng; Zhong, Jun; Xu, Rong [Key Laboratory of Biorheological Science and Technology, Ministry of Education, and Bioengineering College, Chongqing University, Shapingba, Chongqing 400030 (China); Mao, Lizheng [Jiangsu Asialand Biomed-Technology Co. Ltd., Changzhou, Jiangsu 213164 (China); Deng, Linhong, E-mail: dlh@cczu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, and Bioengineering College, Chongqing University, Shapingba, Chongqing 400030 (China); Changzhou Key Laboratory of Respiratory Medical Engineering, Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, Jiangsu 213164 (China)

2016-11-15

A disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptibility gene for asthma, but details of the causality are not fully understood. We hypothesize that soluble ADAM33 (sADAM33) overexpression can alter the mechanical behaviors of airway smooth muscle cells (ASMCs) via regulation of the cell's contractile phenotype, and thus contributes to airway hyperresponsiveness (AHR) in asthma. To test this hypothesis, we either overexpressed or knocked down the sADAM33 in rat ASMCs by transfecting the cells with sADAM33 coding sequence or a small interfering RNA (siRNA) that specifically targets the ADAM33 disintegrin domain, and subsequently assessed the cells for stiffness, contractility and traction force, together with the expression level of contractile and proliferative phenotype markers. We also investigated whether these changes were dependent on Rho/ROCK pathway by culturing the ASMCs either in the absence or presence of ROCK inhibitor (H1152). The results showed that the ASMCs with sADAM33 overexpression were stiffer and more contractile, generated greater traction force, exhibited increased expression levels of contractile phenotype markers and markedly enhanced Rho activation. Furthermore these changes were largely attenuated when the cells were cultured in the presence of H-1152. However, the knock-down of ADAM33 seemed insufficient to influence majority of the mechanical behaviors of the ASMCs. Taken together, we demonstrated that sADAM33 overexpression altered the mechanical behaviors of ASMCs in vitro, which was most likely by promoting a hypercontractile phenotype transition of ASMCs through Rho/ROCK pathway. This revelation may establish the previously missing link between ADAM33 expression and AHR, and also provide useful insight for targeting sADAM33 in asthma prevention and therapy. - Highlights: • sADAM33 overexpression enhances the stiffness, traction force and contractility of ASMCs. • sADAM33 overexpression promotes

Overexpression of soluble ADAM33 promotes a hypercontractile phenotype of the airway smooth muscle cell in rat

International Nuclear Information System (INIS)

Duan, Yiyuan; Long, Jiaoyue; Chen, Jun; Jiang, Xuemei; Zhu, Jian; Jin, Yang; Lin, Feng; Zhong, Jun; Xu, Rong; Mao, Lizheng; Deng, Linhong

2016-01-01

A disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptibility gene for asthma, but details of the causality are not fully understood. We hypothesize that soluble ADAM33 (sADAM33) overexpression can alter the mechanical behaviors of airway smooth muscle cells (ASMCs) via regulation of the cell's contractile phenotype, and thus contributes to airway hyperresponsiveness (AHR) in asthma. To test this hypothesis, we either overexpressed or knocked down the sADAM33 in rat ASMCs by transfecting the cells with sADAM33 coding sequence or a small interfering RNA (siRNA) that specifically targets the ADAM33 disintegrin domain, and subsequently assessed the cells for stiffness, contractility and traction force, together with the expression level of contractile and proliferative phenotype markers. We also investigated whether these changes were dependent on Rho/ROCK pathway by culturing the ASMCs either in the absence or presence of ROCK inhibitor (H1152). The results showed that the ASMCs with sADAM33 overexpression were stiffer and more contractile, generated greater traction force, exhibited increased expression levels of contractile phenotype markers and markedly enhanced Rho activation. Furthermore these changes were largely attenuated when the cells were cultured in the presence of H-1152. However, the knock-down of ADAM33 seemed insufficient to influence majority of the mechanical behaviors of the ASMCs. Taken together, we demonstrated that sADAM33 overexpression altered the mechanical behaviors of ASMCs in vitro, which was most likely by promoting a hypercontractile phenotype transition of ASMCs through Rho/ROCK pathway. This revelation may establish the previously missing link between ADAM33 expression and AHR, and also provide useful insight for targeting sADAM33 in asthma prevention and therapy. - Highlights: • sADAM33 overexpression enhances the stiffness, traction force and contractility of ASMCs. • sADAM33 overexpression promotes a

A deterministic model predicts the properties of stochastic calcium oscillations in airway smooth muscle cells.

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Cao, Pengxing; Tan, Xiahui; Donovan, Graham; Sanderson, Michael J; Sneyd, James

2014-08-01

The inositol trisphosphate receptor ([Formula: see text]) is one of the most important cellular components responsible for oscillations in the cytoplasmic calcium concentration. Over the past decade, two major questions about the [Formula: see text] have arisen. Firstly, how best should the [Formula: see text] be modeled? In other words, what fundamental properties of the [Formula: see text] allow it to perform its function, and what are their quantitative properties? Secondly, although calcium oscillations are caused by the stochastic opening and closing of small numbers of [Formula: see text], is it possible for a deterministic model to be a reliable predictor of calcium behavior? Here, we answer these two questions, using airway smooth muscle cells (ASMC) as a specific example. Firstly, we show that periodic calcium waves in ASMC, as well as the statistics of calcium puffs in other cell types, can be quantitatively reproduced by a two-state model of the [Formula: see text], and thus the behavior of the [Formula: see text] is essentially determined by its modal structure. The structure within each mode is irrelevant for function. Secondly, we show that, although calcium waves in ASMC are generated by a stochastic mechanism, [Formula: see text] stochasticity is not essential for a qualitative prediction of how oscillation frequency depends on model parameters, and thus deterministic [Formula: see text] models demonstrate the same level of predictive capability as do stochastic models. We conclude that, firstly, calcium dynamics can be accurately modeled using simplified [Formula: see text] models, and, secondly, to obtain qualitative predictions of how oscillation frequency depends on parameters it is sufficient to use a deterministic model.

Stimulation of aortic smooth muscle cell mitogenesis by serotonin

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Nemecek, G.M.; Coughlin, S.R.; Handley, D.A.; Moskowitz, M.A.

1986-01-01

Bovine aortic smooth muscle cells in vitro responded to 1 nM to 10 μM serotonin with increased incorporation of [ 3 H]thymidine into DNA. The mitogenic effect of serotonin was half-maximal at 80 nM and maximal above 1 μM. At a concentration of 1 μM, serotonin stimulated smooth muscle cell mitogenesis to the same extent as human platelet-derived growth factor (PDGF) at 12 ng/ml. Tryptamine was ≅ 1/10th as potent as serotonin as a mitogen for smooth muscle cells. Other indoles that are structurally related to serotonin (D- and L-tryptophan, 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, melatonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol) and quipazine were inactive. The stimulatory effect of serotonin on smooth muscle cell DNA synthesis required prolonged (20-24 hr) exposure to the agonist and was attenuated in the presence of serotonin D receptor antagonists. When smooth muscle cells were incubated with submaximal concentrations of serotonin and PDGF, synergistic rather than additive mitogenic responses were observed. These data indicate that serotonin has a significant mitogenic effect on smooth muscle cells in vitro, which appears to be mediated by specific plasma membrane receptors

Expression of smooth muscle and non-muscle myosin heavy chain isoforms in cultured vascular smooth muscle cells

International Nuclear Information System (INIS)

Rovner, A.S.; Murphy, R.A.; Owens, G.K.

1986-01-01

Immunocytochemical studies of cultured smooth muscle cells (SMCs) have disagreed on the nature of myosin expression. This investigation was undertaken to test for the presence of heterogeneous myosin heavy chain (MHC) isoforms in cell culture as a possible explanation for these results. Previously, Rovner et al. detected two MHCs in intact smooth muscles which differed in molecular weight by ca. 4000 daltons (SM1 and SM2) using a 3-4% acrylamide gradient SDS gel system. When sub-confluent primary cultures of rat aorta SMCs were assayed by this system, SM1 and SM2 were seen, along with large amounts of a third, unique MHC, NM, which closely resembled the MHC from human platelet in size and antigenicity. Data from 35 S-methionine autoradiograms showed that the log growth phase SMC cultures were producing almost exclusively NM, but the growth arrest, post-confluent cultures synthesized increased relative amounts of the SM MHC forms and contained comparable amounts of SM1, SM2, and NM. The same patterns of MHC synthesis were seen in sub-passaged SMCs. The expression of the SM-specific forms of myosin in quiescent, post-confluent cultures parallels that of smooth muscle actin suggesting that density induced growth arrest promotes cytodifferentiation in cultured vascular SMCs

Vardenafil inhibiting parasympathetic function of tracheal smooth muscle.

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Lee, Fei-Peng; Chao, Pin-Zhir; Wang, Hsing-Won

2018-07-01

Levitra, a phosphodiesterase-5 (PDE5) inhibitor, is the trade name of vardenafil. Nowadays, it is applied to treatment of erectile dysfunction. PDE5 inhibitors are employed to induce dilatation of the vascular smooth muscle. The effect of Levitra on impotency is well known; however, its effect on the tracheal smooth muscle has rarely been explored. When administered for sexual symptoms via oral intake or inhalation, Levitra might affect the trachea. This study assessed the effects of Levitra on isolated rat tracheal smooth muscle by examining its effect on resting tension of tracheal smooth muscle, contraction caused by 10 -6  M methacholine as a parasympathetic mimetic, and electrically induced tracheal smooth muscle contractions. The results showed that adding methacholine to the incubation medium caused the trachea to contract in a dose-dependent manner. Addition of Levitra at doses of 10 -5  M or above elicited a significant relaxation response to 10 -6  M methacholine-induced contraction. Levitra could inhibit electrical field stimulation-induced spike contraction. It alone had minimal effect on the basal tension of the trachea as the concentration increased. High concentrations of Levitra could inhibit parasympathetic function of the trachea. Levitra when administered via oral intake might reduce asthma attacks in impotent patients because it might inhibit parasympathetic function and reduce methacholine-induced contraction of the tracheal smooth muscle. Copyright © 2018. Published by Elsevier Taiwan LLC.

Immunomodulation of afferent neurons in guinea-pig isolated airway.

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Riccio, M M; Myers, A C; Undem, B J

1996-03-01

1. The trachea, larynx and main bronchi with the right vagus nerve and nodose ganglion were isolated from guinea-pigs passively immunized 24 h previously with serum containing anti-ovalbumin antibody. 2. The airways were placed in one compartment of a Perspex chamber for recording of isometric tension while the nodose ganglion and attached vagus nerve were pulled into another compartment. Action potentials arriving from single airway afferent nerve endings were monitored extracellularly using a glass microelectrode positioned near neuronal cell bodies in the ganglion. Mechanosensitivity of the nerve endings was quantified using calibrated von Frey filaments immediately before and after exposure to antigen (10 micrograms ml-1 ovalbumin). 3. Ten endings responded to the force exerted by the lowest filament (0.078 mN) and were not further investigated. In airways from thirteen immunized guinea-pigs, the mechanical sensitivity of A delta afferent fibres (conduction velocity = 4.3 +/- 0.6 m s-1) was enhanced 4.1 +/- 0.9-fold following airway exposure to antigen (P action potential generation except in one instance when the receptive field was located over the smooth muscle. This ending also responded to methacholine suggesting that spatial changes in the receptive field, induced by muscle contraction, were responsible for the activation. 5. The mediators responsible for these effects are unknown, although histamine, prostaglandins, leukotrienes and tachykinins do not appear to be essential. The increase in mechanical responsiveness was not associated with the smooth muscle contraction since leukotriene C4, histamine and tachykinins, which all caused a similar contraction to antigen, did not affect mechanical thresholds. Moreover, the antigen-induced increases in excitability persisted beyond the duration of the smooth muscle contraction. 6. These results demonstrate that antigen-antibody-mediated inflammatory processes may enhance the excitability of vagal afferent

Effects of Flavin7 on allergen induced hyperreactivity of airways

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Franova S

2009-12-01

Full Text Available Abstract Some studies have suggested that the polyphenolic compounds might reduce the occurrence of asthma symptoms. The aim of our experiments was to evaluate the effects of 21 days of the flavonoid Flavin7 administration on experimentally induced airway inflammation in ovalbumin-sensitized guinea pigs. We assessed tracheal smooth muscle reactivity by an in vitro muscle-strip method; changes in airway resistance by an in vivo plethysmographic method; histological picture of tracheal tissue; and the levels of interleukin 4 (IL-4, and interleukin 5 (IL-5 in bronchoalveolar lavage fluid (BALF. Histological investigation of tracheal tissue and the concentrations of the inflammatory cytokines IL-4 and IL-5 in BALF were used as indices of airway inflammation. Administration of Flavin7 caused a significant decrease of specific airway resistance after histamine nebulization and a decline in tracheal smooth muscle contraction amplitude in response to bronchoconstricting mediators. Flavin7 minimized the degree of inflammation estimated on the basis of eosinophil calculation and IL-4 and IL-5 concentrations. In conclusion, administration of Flavin7 showed bronchodilating and anti-inflammatory effects on allergen-induced airway inflammation.

L-ornithine derived polyamines in cystic fibrosis airways.

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Hartmut Grasemann

Full Text Available Increased arginase activity contributes to airway nitric oxide (NO deficiency in cystic fibrosis (CF. Whether down-stream products of arginase activity contribute to CF lung disease is currently unknown. The objective of this study was to test whether L-ornithine derived polyamines are present in CF airways and contribute to airway pathophysiology. Polyamine concentrations were measured in sputum of patients with CF and in healthy controls, using liquid chromatography-tandem mass spectrometry. The effect of spermine on airway smooth muscle mechanical properties was assessed in bronchial segments of murine airways, using a wire myograph. Sputum polyamine concentrations in stable CF patients were similar to healthy controls for putrescine and spermidine but significantly higher for spermine. Pulmonary exacerbations were associated with an increase in sputum and spermine levels. Treatment for pulmonary exacerbations resulted in decreases in arginase activity, L-ornithine and spermine concentrations in sputum. The changes in sputum spermine with treatment correlated significantly with changes in L-ornithine but not with sputum inflammatory markers. Incubation of mouse bronchi with spermine resulted in an increase in acetylcholine-induced force and significantly reduced nitric oxide-induced bronchial relaxation. The polyamine spermine is increased in CF airways. Spermine contributes to airways obstruction by reducing the NO-mediated smooth muscle relaxation.

Myosin light chain kinase phosphorylation in tracheal smooth muscle

International Nuclear Information System (INIS)

Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E.

1990-01-01

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32 P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+

Importance of contraction history on muscle force of porcine urinary bladder smooth muscle.

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Menzel, Robin; Böl, Markus; Siebert, Tobias

2017-02-01

The purpose of this study was to provide a comprehensive dataset of porcine urinary bladder smooth muscle properties. Particularly, the history dependence of force production, namely force depression (FD) following shortening and force enhancement (FE) following stretch, was analysed. During active micturition, the circumference of the urinary bladder changes enormously. Thus, FD might be an important phenomenon during smooth muscle contraction. Electrically stimulated, intact urinary bladder strips from pigs (n = 10) were suspended in an aerated-filled organ bath, and different isometric, isotonic, and isokinetic contraction protocols were performed to determine the force-length and the force-velocity relation. FD and FE were assessed in concentric and eccentric contractions with different ramp lengths and ramp velocities. Bladder smooth muscles exhibit considerable amounts of FD and FE. The amount of FD increased significantly with ramp length, while FE did not change. However, FE and FD were independent of ramp velocity. The results imply that smooth muscle bladder strips exhibit similar muscle properties and history-dependent behaviour compared to striated muscles. The provided dataset of muscle properties is important for bladder modelling as well as for the analyses and interpretation of dynamic bladder filling and voiding.

Ureter smooth muscle cell orientation in rat is predominantly longitudinal.

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Bart Spronck

Full Text Available In ureter peristalsis, the orientation of the contracting smooth muscle cells is essential, yet current descriptions of orientation and composition of the smooth muscle layer in human as well as in rat ureter are inconsistent. The present study aims to improve quantification of smooth muscle orientation in rat ureters as a basis for mechanistic understanding of peristalsis. A crucial step in our approach is to use two-photon laser scanning microscopy and image analysis providing objective, quantitative data on smooth muscle cell orientation in intact ureters, avoiding the usual sectioning artifacts. In 36 rat ureter segments, originating from a proximal, middle or distal site and from a left or right ureter, we found close to the adventitia a well-defined longitudinal smooth muscle orientation. Towards the lamina propria, the orientation gradually became slightly more disperse, yet the main orientation remained longitudinal. We conclude that smooth muscle cell orientation in rat ureter is predominantly longitudinal, though the orientation gradually becomes more disperse towards the proprial side. These findings do not support identification of separate layers. The observed longitudinal orientation suggests that smooth muscle contraction would rather cause local shortening of the ureter, than cause luminal constriction. However, the net-like connective tissue of the ureter wall may translate local longitudinal shortening into co-local luminal constriction, facilitating peristalsis. Our quantitative, minimally invasive approach is a crucial step towards more mechanistic insight into ureter peristalsis, and may also be used to study smooth muscle cell orientation in other tube-like structures like gut and blood vessels.

Ureter smooth muscle cell orientation in rat is predominantly longitudinal.

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Spronck, Bart; Merken, Jort J; Reesink, Koen D; Kroon, Wilco; Delhaas, Tammo

2014-01-01

In ureter peristalsis, the orientation of the contracting smooth muscle cells is essential, yet current descriptions of orientation and composition of the smooth muscle layer in human as well as in rat ureter are inconsistent. The present study aims to improve quantification of smooth muscle orientation in rat ureters as a basis for mechanistic understanding of peristalsis. A crucial step in our approach is to use two-photon laser scanning microscopy and image analysis providing objective, quantitative data on smooth muscle cell orientation in intact ureters, avoiding the usual sectioning artifacts. In 36 rat ureter segments, originating from a proximal, middle or distal site and from a left or right ureter, we found close to the adventitia a well-defined longitudinal smooth muscle orientation. Towards the lamina propria, the orientation gradually became slightly more disperse, yet the main orientation remained longitudinal. We conclude that smooth muscle cell orientation in rat ureter is predominantly longitudinal, though the orientation gradually becomes more disperse towards the proprial side. These findings do not support identification of separate layers. The observed longitudinal orientation suggests that smooth muscle contraction would rather cause local shortening of the ureter, than cause luminal constriction. However, the net-like connective tissue of the ureter wall may translate local longitudinal shortening into co-local luminal constriction, facilitating peristalsis. Our quantitative, minimally invasive approach is a crucial step towards more mechanistic insight into ureter peristalsis, and may also be used to study smooth muscle cell orientation in other tube-like structures like gut and blood vessels.

Modeling the dispersion effects of contractile fibers in smooth muscles

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Murtada, Sae-Il; Kroon, Martin; Holzapfel, Gerhard A.

2010-12-01

Micro-structurally based models for smooth muscle contraction are crucial for a better understanding of pathological conditions such as atherosclerosis, incontinence and asthma. It is meaningful that models consider the underlying mechanical structure and the biochemical activation. Hence, a simple mechanochemical model is proposed that includes the dispersion of the orientation of smooth muscle myofilaments and that is capable to capture available experimental data on smooth muscle contraction. This allows a refined study of the effects of myofilament dispersion on the smooth muscle contraction. A classical biochemical model is used to describe the cross-bridge interactions with the thin filament in smooth muscles in which calcium-dependent myosin phosphorylation is the only regulatory mechanism. A novel mechanical model considers the dispersion of the contractile fiber orientations in smooth muscle cells by means of a strain-energy function in terms of one dispersion parameter. All model parameters have a biophysical meaning and may be estimated through comparisons with experimental data. The contraction of the middle layer of a carotid artery is studied numerically. Using a tube the relationships between the internal pressure and the stretches are investigated as functions of the dispersion parameter, which implies a strong influence of the orientation of smooth muscle myofilaments on the contraction response. It is straightforward to implement this model in a finite element code to better analyze more complex boundary-value problems.

Cardiac, Skeletal, and smooth muscle mitochondrial respiration

DEFF Research Database (Denmark)

Park, Song-Young; Gifford, Jayson R; Andtbacka, Robert H I

2014-01-01

, skeletal, and smooth muscle was harvested from a total of 22 subjects (53±6 yrs) and mitochondrial respiration assessed in permeabilized fibers. Complex I+II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac, skeletal, to smooth muscle (54±1; 39±4; 15......±1 pmol•s(-1)•mg (-1), prespiration rates were normalized by CS (respiration...... per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, Complex I state 2 normalized for CS activity, an index of non-phosphorylating respiration per mitochondrial content, increased progressively from cardiac, skeletal...

PPARγ ligand ciglitazone inhibits TNFα-induced ICAM-1 in human airway smooth muscle cells

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Chien-Da Huang

2014-08-01

Full Text Available Background: Modification of human airway smooth muscle (ASM function by proinflammatory cytokines has been regarded as a potential mechanism underlying bronchial hyperresponsiveness in asthma. Human ASM cells express intercellular adhesion molecule (ICAM-1 in response to cytokines. Synthetic ligands for peroxisome proliferator-activated receptor (PPARγ reportedly possess anti-inflammatory and immunomodulatory properties. In this study, we examined whether ciglitazone, a synthetic PPARγ ligand, can modulate the basal and tumor necrosis factor (TNFα-induced ICAM1 gene expression in human ASM cells. Methods: Human ASM cells were treated with TNFα. ICAM-1 expression was assessed by flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR analysis. PPARγ activity was inhibited by target-specific small interfering (si RNA targeting PPARγ and GW9662, a PPARγ antagonist. Activity of nuclear factor (NF-κB was assessed by using immunoblot analysis, immune-confocal images, and electrophoretic mobility shift assay (EMSA. Results: By flow cytometry, ciglitazone alone had no effect on ICAM-1 expression in ASM cells, but inhibited ICAM-1 expression in response to TNFα (10 ng/ml in a dose-dependent manner (1-10 μM. It also inhibited TNFα-induced ICAM1 gene expression by RT-PCR analysis. Knockdown of PPARγ gene by target-specific siRNA targeting PPARγ enhanced ICAM-1 expression and the inhibitory effect of ciglitazone on TNFα-induced ICAM-1 expression was reversed by PPARγ siRNA and GW9662. SN-50 (10 μg/ml, an inhibitor for nuclear translocation of NF-κB, inhibited TNFα-induced ICAM-1 expression. Ciglitazone did not prevent TNFα-induced degradation of the cytosolic inhibitor of NF-κB (IκB, but inhibited the nuclear translocation of p65 induced by TNFα and suppressed the NF-κB/DNA binding activity. Conclusion: These findings suggest that ciglitazone inhibits TNFα-induced ICAM1 gene expression in human ASM cells through

Markers of Airway Remodeling in Bronchopulmonary Diseases

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O.Ye. Chernyshova

2014-10-01

Full Text Available The article presents information about markers of airway remodeling in bronchopulmonary diseases. There is described the influence of matrix metalloproteinases, tissue inhibitor of matrix metalloproteinase, transforming growth factor, collagen autoantibodies III type, endothelin-1 on the processes of morphological airway reconstruction as smooth muscle hypertrophy, enhanced neovascularization, epithelial cell hyperplasia, collagen deposition, compaction of the basal membrane, observed in bronchial asthma.

Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

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Yun-Yun Ma

Full Text Available Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification.

Research Upregulation of CD23 (FcεRII Expression in Human Airway Smooth Muscle Cells (huASMC in Response to IL-4, GM-CSF, and IL-4/GM-CSF

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Lew D Betty

2005-05-01

Full Text Available Abstract Background Airway smooth muscle cells play a key role in remodeling that contributes to airway hyperreactivity. Airway smooth muscle remodeling includes hypertrophy and hyperplasia. It has been previously shown that the expression of CD23 on ASMC in rabbits can be induced by the IgE component of the atopic serum. We examined if other components of atopic serum are capable of inducing CD23 expression independent of IgE. Methods Serum starved huASMC were stimulated with either IL-4, GM-CSF, IL-13, IL-5, PGD2, LTD4, tryptase or a combination of IL-4, IL-5, IL-13 each with GM-CSF for a period of 24 h. CD23 expression was analyzed by flow cytometry, western blot, and indirect immunofluorescence. Results The CD23 protein expression was upregulated in huASMC in response to IL-4, GM-CSF, and IL-4/GM-CSF. The percentage of cells with increased fluorescence intensity above the control was 25.1 ± 4.2% (IL-4, 15.6 ± 2.7% (GM-CSF and 32.9 ± 13.9% (IL-4/GMCSF combination(n = 3. The protein content of IL-4/GMCSF stimulated cells was significantly elevated. Expression of CD23 in response to IL-4, GM-CSF, IL-4/GM-CSF was accompanied by changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling. Western blot revealed abundant expression of the IL-4Rα and a low level expression of IL-2Rγc in huASMC. Stimulation with IL-4 resulted in the phosphorylation of STAT-6 and an increase in the expression of the IL-2Rγc. Conclusion CD23 on huASMC is upregulated by IL-4, GM-CSF, and IL-4/GM-CSF. The expression of CD23 is accompanied by an increase in cell volume and an increase in protein content per cell, suggesting hypertrophy. Upregulation of CD23 by IL-4/GM-CSF results in phenotypic changes in huASMC that could play a role in cell migration or a change in the synthetic function of the cells. Upregulation of CD23 in huASMC by IL-4 and GM-CSF can contribute to changes in huASMC and may provide an avenue

Effect of parenchymal stiffness on canine airway size with lung inflation.

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Robert H Brown

2010-04-01

Full Text Available Although airway patency is partially maintained by parenchymal tethering, this structural support is often ignored in many discussions of asthma. However, agonists that induce smooth muscle contraction also stiffen the parenchyma, so such parenchymal stiffening may serve as a defense mechanism to prevent airway narrowing or closure. To quantify this effect, specifically how changes in parenchymal stiffness alter airway size at different levels of lung inflation, in the present study, we devised a method to separate the effect of parenchymal stiffening from that of direct airway narrowing. Six anesthetized dogs were studied under four conditions: baseline, after whole lung aerosol histamine challenge, after local airway histamine challenge, and after complete relaxation of the airways. In each of these conditions, we used High resolution Computed Tomography to measure airway size and lung volume at five different airway pressures (0, 12, 25, 32, and 45 cm H(2O. Parenchymal stiffening had a protective effect on airway narrowing, a fact that may be important in the airway response to deep inspiration in asthma. When the parenchyma was stiffened by whole lung aerosol histamine challenge, at every lung volume above FRC, the airways were larger than when they were directly challenged with histamine to the same initial constriction. These results show for the first time that a stiff parenchyma per se minimizes the airway narrowing that occurs with histamine challenge at any lung volume. Thus in clinical asthma, it is not simply increased airway smooth muscle contraction, but perhaps a lack of homogeneous parenchymal stiffening that contributes to the symptomatic airway hyperresponsiveness.

Overexpression of microRNA-375 impedes platelet-derived growth factor-induced proliferation and migration of human fetal airway smooth muscle cells by targeting Janus kinase 2.

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Ji, Yamei; Yang, Xin; Su, Huixia

2018-02-01

The abnormal proliferation and migration of airway smooth muscle (ASM) cells play a critical role in airway remodeling during the development of asthma. MicroRNAs (miRNAs) have emerged as critical regulators of ASM cell proliferation and migration in airway remodeling. In this study, we aimed to investigate the potential role of miR-375 in the regulation of platelet-derived growth factor (PDGF)-induced fetal ASM cell proliferation and migration. Our results showed that miR-375 expression was significantly decreased in fetal ASM cells that were treated with PDGF. Functional data showed that overexpression of miR-375 inhibited the proliferation and migration of fetal ASM cells, whereas inhibition of miR-375 enhanced the proliferation and migration of fetal ASM cells. The results of bioinformatics analysis and a dual-luciferase reporter assay showed that miR-375 binds directly to the 3'-untranslated region of Janus kinase 2 (JAK2). Further data confirmed that miR-375 negatively regulates the expression of JAK2 in fetal ASM cells. Moreover, miR-375 also impeded the PDGF-induced activation of signal transducer and activator of transcription 3 (STAT3) in fetal ASM cells. However, restoration of JAK2 expression partially reversed the inhibitory effect of miR-375 on fetal ASM cell proliferation and migration. Overall, our results demonstrate that miR-375 inhibits fetal ASM cell proliferation and migration by targeting JAK2/STAT3 signaling. Our study provides a potential therapeutic target for the development of novel treatment strategies for pediatric asthma. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Cardiac, skeletal, and smooth muscle mitochondrial respiration: are all mitochondria created equal?

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Park, Song-Young; Gifford, Jayson R; Andtbacka, Robert H I; Trinity, Joel D; Hyngstrom, John R; Garten, Ryan S; Diakos, Nikolaos A; Ives, Stephen J; Dela, Flemming; Larsen, Steen; Drakos, Stavros; Richardson, Russell S

2014-08-01

Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s(-1)·mg(-1), P respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s(-1)·mg(-1), P respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production.

The persistence of active smooth muscle in the female rat cervix through pregnancy.

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Ferland, David J; Darios, Emma S; Watts, Stephanie W

2015-02-01

A controversy exists as to whether functional smooth muscle exists in the cervix before and during pregnancy, potentially continuous with the uterus. We hypothesized that cervical smooth muscle persists through pregnancy and is functional. Uteri and cervices were taken from female virgin, 11 day, and 20 day (near labor) pregnant rats. All experiments used the uterus as a positive control. Three different smooth muscle proteins (smooth muscle α-actin, SM-22α, and calponin-1) allowed immunohistochemical detection of the continuous nature of the smooth muscle from the vagina, cervix, and uterus. Tissues were also hung in isolated tissue baths for the measurement of isometric smooth muscle contraction. Uterine and cervical homogenates were also used in Western analyses to measure protein expression. Immunohistochemistry revealed there to be smooth muscle as validated by an expression of all 3 markers in the cervix. This smooth muscle was continuous with that of the vagina and uterus. Smooth muscle α-actin was detected in virgin tissue (291.3 ± 32.2 arbitrary densitometry units/β-actin), day 11 (416.8 ± 19.5), and day 20 pregnant tissue (293.0 ± 34.4). The virgin, day 11, and day 20 cervices contracted 2.18 ± 0.24 g, 1.46 ± 0.08 g, and 3.88 ± 0.49 g (respectively) to depolarizing KCl. Cervices contracted at day 20 to the cholinergic muscarinic agonist carbamylcholine (maximum, 133% ± 18.2% KCl contraction, n = 4). These findings strongly support that smooth muscle is present in the cervix through pregnancy and continuous with the uterus. Copyright © 2015 Elsevier Inc. All rights reserved.

Calcium signaling in smooth muscle.

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Hill-Eubanks, David C; Werner, Matthias E; Heppner, Thomas J; Nelson, Mark T

2011-09-01

Changes in intracellular Ca(2+) are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca(2+) signal is a reflection of the source of Ca(2+) (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca(2+) entry may be detected optically as graded elevations in intracellular Ca(2+), junctional Ca(2+) transients, Ca(2+) flashes, or Ca(2+) sparklets, whereas release of Ca(2+) from intracellular stores may manifest as Ca(2+) sparks, Ca(2+) puffs, or Ca(2+) waves. These diverse Ca(2+) signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).

An α-smooth muscle actin (acta2/αsma zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells.

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Thomas R Whitesell

Full Text Available Mural cells of the vascular system include vascular smooth muscle cells (SMCs and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of mural cell development in vertebrates is α smooth muscle actin (acta2; αsma, which is expressed by pericytes and SMCs. In vivo models of vascular mural cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing cells. These transgenic fish were used to trace the live development of mural cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive cells and the number of types of blood vessels associated with mural cells increases. Interestingly, the mural cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish mural cells develop relatively late, and have little mobility once they associate with vessels.

Sex, stress and sleep apnoea: Decreased susceptibility to upper airway muscle dysfunction following intermittent hypoxia in females.

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O'Halloran, Ken D; Lewis, Philip; McDonald, Fiona

2017-11-01

Obstructive sleep apnoea syndrome (OSAS) is a devastating respiratory control disorder more common in men than women. The reasons for the sex difference in prevalence are multifactorial, but are partly attributable to protective effects of oestrogen. Indeed, OSAS prevalence increases in post-menopausal women. OSAS is characterized by repeated occlusions of the pharyngeal airway during sleep. Dysfunction of the upper airway muscles controlling airway calibre and collapsibility is implicated in the pathophysiology of OSAS, and sex differences in the neuro-mechanical control of upper airway patency are described. It is widely recognized that chronic intermittent hypoxia (CIH), a cardinal feature of OSAS due to recurrent apnoea, drives many of the morbid consequences characteristic of the disorder. In rodents, exposure to CIH-related redox stress causes upper airway muscle weakness and fatigue, associated with mitochondrial dysfunction. Of interest, in adults, there is female resilience to CIH-induced muscle dysfunction. Conversely, exposure to CIH in early life, results in upper airway muscle weakness equivalent between the two sexes at 3 and 6 weeks of age. Ovariectomy exacerbates the deleterious effects of exposure to CIH in adult female upper airway muscle, an effect partially restored by oestrogen replacement therapy. Intriguingly, female advantage intrinsic to upper airway muscle exists with evidence of substantially greater loss of performance in male muscle during acute exposure to severe hypoxic stress. Sex differences in upper airway muscle physiology may have relevance to human OSAS. The oestrogen-oestrogen receptor α axis represents a potential therapeutic target in OSAS, particularly in post-menopausal women. Copyright © 2016 Elsevier B.V. All rights reserved.

Increased IGF-IEc expression and mechano-growth factor production in intestinal muscle of fibrostenotic Crohn's disease and smooth muscle hypertrophy.

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Li, Chao; Vu, Kent; Hazelgrove, Krystina; Kuemmerle, John F

2015-12-01

The igf1 gene is alternatively spliced as IGF-IEa and IGF-IEc variants in humans. In fibrostenotic Crohn's disease, the fibrogenic cytokine TGF-β1 induces IGF-IEa expression and IGF-I production in intestinal smooth muscle and results in muscle hyperplasia and collagen I production that contribute to stricture formation. Mechano-growth factor (MGF) derived from IGF-IEc induces skeletal and cardiac muscle hypertrophy following stress. We hypothesized that increased IGF-IEc expression and MGF production mediated smooth muscle hypertrophy also characteristic of fibrostenotic Crohn's disease. IGF-IEc transcripts and MGF protein were increased in muscle cells isolated from fibrostenotic intestine under regulation by endogenous TGF-β1. Erk5 and MEF2C were phosphorylated in vivo in fibrostenotic muscle; both were phosphorylated and colocalized to nucleus in response to synthetic MGF in vitro. Smooth muscle-specific protein expression of α-smooth muscle actin, γ-smooth muscle actin, and smoothelin was increased in affected intestine. Erk5 inhibition or MEF2C siRNA blocked smooth muscle-specific gene expression and hypertrophy induced by synthetic MGF. Conditioned media of cultured fibrostenotic muscle induced muscle hypertrophy that was inhibited by immunoneutralization of endogenous MGF or pro-IGF-IEc. The results indicate that TGF-β1-dependent IGF-IEc expression and MGF production in patients with fibrostenotic Crohn's disease regulates smooth muscle cell hypertrophy a critical factor that contributes to intestinal stricture formation. Copyright © 2015 the American Physiological Society.

Chronic respiratory aeroallergen exposure in mice induces epithelial-mesenchymal transition in the large airways.

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Jill R Johnson

Full Text Available Chronic allergic asthma is characterized by Th2-polarized inflammation and leads to airway remodeling and fibrosis but the mechanisms involved are not clear. To determine whether epithelial-mesenchymal transition contributes to airway remodeling in asthma, we induced allergic airway inflammation in mice by intranasal administration of house dust mite (HDM extract for up to 15 consecutive weeks. We report that respiratory exposure to HDM led to significant airway inflammation and thickening of the smooth muscle layer in the wall of the large airways. Transforming growth factor beta-1 (TGF-β1 levels increased in mouse airways while epithelial cells lost expression of E-cadherin and occludin and gained expression of the mesenchymal proteins vimentin, alpha-smooth muscle actin (α-SMA and pro-collagen I. We also observed increased expression and nuclear translocation of Snail1, a transcriptional repressor of E-cadherin and a potent inducer of EMT, in the airway epithelial cells of HDM-exposed mice. Furthermore, fate-mapping studies revealed migration of airway epithelial cells into the sub-epithelial regions of the airway wall. These results show the contribution of EMT to airway remodeling in chronic asthma-like inflammation and suggest that Th2-polarized airway inflammation can trigger invasion of epithelial cells into the subepithelial regions of the airway wall where they contribute to fibrosis, demonstrating a previously unknown plasticity of the airway epithelium in allergic airway disease.

CysLT1 receptor-induced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation

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Capra Valérie

2006-03-01

Full Text Available Abstract Background Cysteine-containing leukotrienes (cysteinyl-LTs are pivotal inflammatory mediators that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. In particular, cysteinyl-LTs exert a variety of effects with relevance to the aetiology of asthma such as smooth muscle contraction, eosinophil recruitment, increased microvascular permeability, enhanced mucus secretion and decreased mucus transport and, finally, airway smooth muscle cells (ASMC proliferation. We used human ASMC (HASMC to identify the signal transduction pathway(s of the leukotriene D4 (LTD4-induced DNA synthesis. Methods Proliferation of primary HASMC was measured by [3H]thymidine incorporation. Phosphorylation of EGF receptor (EGF-R and ERK1/2 was assessed with a polyclonal anti-EGF-R or anti-phosphoERKl/2 monoclonal antibody. A Ras pull-down assay kit was used to evaluate Ras activation. The production of reactive oxygen species (ROS was estimated by measuring dichlorodihydrofluorescein (DCF oxidation. Results We demonstrate that in HASMC LTD4-stimulated thymidine incorporation and potentiation of EGF-induced mitogenic signaling mostly depends upon EGF-R transactivation through the stimulation of CysLT1-R. Accordingly, we found that LTD4 stimulation was able to trigger the increase of Ras-GTP and, in turn, to activate ERK1/2. We show here that EGF-R transactivation was sensitive to pertussis toxin (PTX and phosphoinositide 3-kinase (PI3K inhibitors and that it occurred independently from Src activity, despite the observation of a strong impairment of LTD4-induced DNA synthesis following Src inhibition. More interestingly, CysLT1-R stimulation increased the production of ROS and N-acetylcysteine (NAC abolished LTD4-induced EGF-R phosphorylation and thymidine incorporation. Conclusion Collectively, our data demonstrate that in HASMC LTD4 stimulation of a Gi/o coupled CysLT1-R triggers the transactivation of the EGF

Smooth muscle adaptation after intestinal transection and resection.

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Thompson, J S; Quigley, E M; Adrian, T E

1996-09-01

Changes in motor function occur in the intestinal remnant after intestinal resection. Smooth muscle adaptation also occurs, particularly after extensive resection. The time course of these changes and their interrelationship are unclear. Our aim was to evaluate changes in canine smooth muscle structure and function during intestinal adaptation after transection and resection. Twenty-five dogs underwent either transection (N = 10), 50% distal resection (N = 10), or 50% proximal resection (N = 5). Thickness and length of the circular (CM) and longitudinal (LM) muscle layers were measured four and 12 weeks after resection. In vitro length-tension properties and response to a cholinergic agonist were studied in mid-jejunum and mid-ileum. Transection alone caused increased CM length in the jejunum proximal to the transection but did not affect LM length or muscle thickness. A 50% resection resulted in increased length of CM throughout the intestine and thickening of CM and LM near the anastomosis. Active tension of jejunal CM increased transiently four weeks after resection. Active tension in jejunal LM was decreased 12 weeks after transection and resection. Sensitivity of CM to carbachol was similar after transection and resection. It is concluded that: (1) Structural adaptation of both circular and longitudinal muscle occurs after intestinal resection. (2) This process is influenced by the site of the intestinal remnant. (3) Only minor and transient changes occur in smooth muscle function after resection. (4) Factors other than muscle adaptation are likely involved in the changes in motor function seen following massive bowel resection.

Control of upper airway muscle activity in younger versus older men during sleep onset

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Fogel, Robert B; White, David P; Pierce, Robert J; Malhotra, Atul; Edwards, Jill K; Dunai, Judy; Kleverlaan, Darci; Trinder, John

2003-01-01

Pharyngeal dilator muscles are clearly important in the pathophysiology of obstructive sleep apnoea syndrome (OSA). We have previously shown that the activity of both the genioglossus (GGEMG) and tensor palatini (TPEMG) are decreased at sleep onset, and that this decrement in muscle activity is greater in the apnoea patient than in healthy controls. We have also previously shown this decrement to be greater in older men when compared with younger ones. In order to explore the mechanisms responsible for this decrement in muscle activity nasal continuous positive airway pressure (CPAP) was applied to reduce negative pressure mediated muscle activation. We then investigated the effect of sleep onset (transition from predominantly α to predominantly θ EEG activity) on ventilation, upper airway muscle activation and upper airway resistance (UAR) in middle-aged and younger healthy men. We found that both GGEMG and TPEMG were reduced by the application of nasal CPAP during wakefulness, but that CPAP did not alter the decrement in activity in either muscle seen in the first two breaths following an α to θ transition. However, CPAP prevented both the rise in UAR at sleep onset that occurred on the control night, and the recruitment in GGEMG seen in the third to fifth breaths following the α to θ transition. Further, GGEMG was higher in the middle-aged men than in the younger men during wakefulness and was decreased more in the middle-aged men with the application of nasal CPAP. No differences were seen in TPEMG between the two age groups. These data suggest that the initial sleep onset reduction in upper airway muscle activity is due to loss of a ‘wakefulness’ stimulus, rather than to loss of responsiveness to negative pressure. In addition, it suggests that in older men, higher wakeful muscle activity is due to an anatomically more collapsible upper airway with more negative pressure driven muscle activation. Sleep onset per se does not appear to have a greater

Bronchoconstriction Induces TGF-β Release and Airway Remodelling in Guinea Pig Lung Slices.

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Tjitske A Oenema

Full Text Available Airway remodelling, including smooth muscle remodelling, is a primary cause of airflow limitation in asthma. Recent evidence links bronchoconstriction to airway remodelling in asthma. The mechanisms involved are poorly understood. A possible player is the multifunctional cytokine TGF-β, which plays an important role in airway remodelling. Guinea pig lung slices were used as an in vitro model to investigate mechanisms involved in bronchoconstriction-induced airway remodelling. To address this aim, mechanical effects of bronchoconstricting stimuli on contractile protein expression and TGF-β release were investigated. Lung slices were viable for at least 48 h. Both methacholine and TGF-β1 augmented the expression of contractile proteins (sm-α-actin, sm-myosin, calponin after 48 h. Confocal fluorescence microscopy showed that increased sm-myosin expression was enhanced in the peripheral airways and the central airways. Mechanistic studies demonstrated that methacholine-induced bronchoconstriction mediated the release of biologically active TGF-β, which caused the increased contractile protein expression, as inhibition of actin polymerization (latrunculin A or TGF-β receptor kinase (SB431542 prevented the methacholine effects, whereas other bronchoconstricting agents (histamine and KCl mimicked the effects of methacholine. Collectively, bronchoconstriction promotes the release of TGF-β, which induces airway smooth muscle remodelling. This study shows that lung slices are a useful in vitro model to study mechanisms involved in airway remodelling.

IFN-γ, IL-4 and IL-13 modulate responsiveness of human airway smooth muscle cells to IL-13

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Michoud Marie-Claire

2008-12-01

Full Text Available Abstract Background IL-13 is a critical mediator of allergic asthma and associated airway hyperresponsiveness. IL-13 acts through a receptor complex comprised of IL-13Rα1 and IL-4Rα subunits with subsequent activation of signal transducer and activator of transcription 6 (STAT6. The IL-13Rα2 receptor may act as a decoy receptor. In human airway smooth muscle (HASM cells, IL-13 enhances cellular proliferation, calcium responses to agonists and induces eotaxin production. We investigated the effects of pre-treatment with IL-4, IL-13 and IFN-γ on the responses of HASM cells to IL-13. Methods Cultured HASM were examined for expression of IL-13 receptor subunits using polymerase chain reaction, immunofluorescence microscopy and flow cytometry. Effects of cytokine pre-treatment on IL-13-induced cell responses were assessed by looking at STAT6 phosphorylation using Western blot, eotaxin secretion and calcium responses to histamine. Results IL-13Rα1, IL-4Rα and IL-13Rα2 subunits were expressed on HASM cells. IL-13 induced phosphorylation of STAT6 which reached a maximum by 30 minutes. Pre-treatment with IL-4, IL-13 and, to a lesser degree, IFN-γ reduced peak STAT6 phosphorylation in response to IL-13. IL-13, but not IFN-γ, pre-treatment abrogated IL-13-induced eotaxin secretion. Pre-treatment with IL-4 or IL-13 abrogated IL-13-induced augmentation of the calcium transient evoked by histamine. Cytokine pre-treatment did not affect expression of IL-13Rα1 and IL-4Rα but increased expression of IL-13Rα2. An anti-IL-13Rα2 neutralizing antibody did not prevent the cytokine pre-treatment effects on STAT6 phosphorylation. Cytokine pre-treatment increased SOCS-1, but not SOCS-3, mRNA expression which was not associated with significant increases in protein expression. Conclusion Pre-treatment with IL-4 and IL-13, but not IFN-γ, induced desensitization of the HASM cells to IL-13 as measured by eotaxin secretion and calcium transients to histamine

A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin.

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Smith, A F; Bigsby, R M; Word, R A; Herring, B P

1998-05-01

A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from -163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (-2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between -1447 and -1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between -1447 and -1474.

Relationship between airway responsiveness to mannitol and to methacholine and markers of airway inflammation, peak flow variability and quality of life in asthma patients

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Porsbjerg, C.; Brannan, J.D.; Anderson, S.D.

2008-01-01

Background Airway hyperresponsiveness (AHR) to stimuli that cause bronchial smooth muscle (BSM) contraction indirectly through the release of endogenous mediators is thought to reflect air-way inflammation more closely compared with AHR measured by stimuli that act directly on BSM. Methods Fifty......, there was a stronger correlation between AHR to mannitol and the level of eNO [PD15 to mannitol vs. eNO (p.p.b.): r: -0.63, P

Isolation and characterization of the inositol trisphosphate receptor from smooth muscle

International Nuclear Information System (INIS)

Chadwick, C.C.; Saito, A.; Fleischer, S.

1990-01-01

The release of Ca 2+ from internal stores is requisite to muscle contraction. In skeletal muscle and heart, the Ca 2+ release channels (ryanodine receptor) of sarcoplasmic reticulum, involved in excitation-contraction coupling, have recently been isolated and characterized. In smooth muscle, inositol 1,4,5-trisphosphate (IP 3 ) is believed to mobilize Ca 2+ from internal stores and thereby modulate contraction. The authors describe the isolation of an IP 3 receptor from smooth muscle. Bovine aorta smooth muscle microsomes were solubilized with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, and the IP 3 receptor was purified by sucrose gradient centrifugation and column chromatography with heparin-agarose and wheat germ agglutinin-agarose. The receptor is an oligomer of a single polypeptide with a M r of 224,000 as determined by SDS/PAGE. Negative-staining electron microscopy reveals that the receptor is a large pinwheel-like structure having surface dimensions of ∼250 x 250 angstrom with fourfold symmetry. The IP 3 receptor from smooth muscle is similar to the ryanodine receptor with regard to its large size and fourfold symmetry, albeit distinct with regard to appearance, protomer size, and ligand binding

A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABAA Receptors in the Lung.

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Forkuo, Gloria S; Nieman, Amanda N; Kodali, Revathi; Zahn, Nicolas M; Li, Guanguan; Rashid Roni, M S; Stephen, Michael Rajesh; Harris, Ted W; Jahan, Rajwana; Guthrie, Margaret L; Yu, Olivia B; Fisher, Janet L; Yocum, Gene T; Emala, Charles W; Steeber, Douglas A; Stafford, Douglas C; Cook, James M; Arnold, Leggy A

2018-05-07

We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric α 5 β 3 γ 2 selective GABA A receptor (GABA A R) ligands. MIDD0301 relaxed airway smooth muscle at single micromolar concentrations as demonstrated with ex vivo guinea pig tracheal rings. MIDD0301 also attenuated airway hyperresponsiveness (AHR) in an ovalbumin murine model of asthma by oral administration. Reduced numbers of eosinophils and macrophages were observed in mouse bronchoalveolar lavage fluid without changing mucous metaplasia. Importantly, lung cytokine expression of IL-17A, IL-4, and TNF-α were reduced for MIDD0301-treated mice without changing antiinflammatory cytokine IL-10 levels. Automated patch clamp confirmed amplification of GABA induced current mediated by α 1-3,5 β 3 γ 2 GABA A Rs in the presence of MIDD0301. Pharmacodynamically, transmembrane currents of ex vivo CD4 + T cells from asthmatic mice were potentiated by MIDD0301 in the presence of GABA. The number of CD4 + T cells observed in the lung of MIDD0301-treated mice were reduced by an oral treatment of 20 mg/kg b.i.d. for 5 days. A half-life of almost 14 h was demonstrated by pharmacokinetic studies (PK) with no adverse CNS effects when treated mice were subjected to sensorimotor studies using the rotarod. PK studies also confirmed very low brain distribution. In conclusion, MIDD0301 represents a safe and improved oral asthma drug candidate that relaxes airway smooth muscle and attenuates inflammation in the lung leading to a reduction of AHR at a dosage lower than earlier reported GABA A R ligands.

Low density lipoprotein uptake by an endothelial-smooth muscle cell bilayer

International Nuclear Information System (INIS)

Alexander, J.J.; Miguel, R.; Graham, D.

1991-01-01

To study the interaction of endothelial and smooth muscle cells, and the means by which such interaction may affect lipid permeability of the arterial wall, cell bilayers were established by use of a transwell culture system. After confluent growth of both cell types had been achieved, iodine 125 bound to low-density lipoprotein (10 ng protein/ml) was added to the media of the upper well. After a 3-hour incubation period, the iodine 125-bound low-density lipoprotein content of the upper and lower media demonstrated an impedance to lipoprotein movement across the endothelial cell monolayer as compared to the bare porous polycarbonate filter of the transwell (p less than 10(-6)). The presence of smooth muscle cells in the bottom well significantly enhanced the permeability of the endothelial cell layer (p less than 10(-60)). This effect remained unchanged over a 9-day time course. Membrane binding and cellular uptake of low-density lipoprotein by endothelial cells was not altered by smooth muscle cells, indicating that this change in permeability could not be easily attributed to changes in receptor-mediated transport or transcytosis. Membrane binding (p less than 0.02) and cellular uptake (p less than 10(-6)) of low-density lipoprotein by smooth muscle cells in the bilayer, when adjusted for counts available in the smooth muscle cell media, were both reduced in the early incubation period as compared to isolated smooth muscle cells. The disproportionate reduction in uptake as compared to binding would suggest that this was not entirely a receptor-dependent process

Constitutively active signaling by the G protein βγ-subunit mediates intrinsically increased phosphodiesterase-4 activity in human asthmatic airway smooth muscle cells.

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Aihua Hu

Full Text Available Signaling by the Gβγ subunit of Gi protein, leading to downstream c-Src-induced activation of the Ras/c-Raf1/MEK-ERK1/2 signaling pathway and its upregulation of phosphodiesterase-4 (PDE4 activity, was recently shown to mediate the heightened contractility in proasthmatic sensitized isolated airway smooth muscle (ASM, as well as allergen-induced airway hyperresponsiveness and inflammation in an in vivo animal model of allergic asthma. This study investigated whether cultured human ASM (HASM cells derived from asthmatic donor lungs exhibit constitutively increased PDE activity that is attributed to intrinsically upregulated Gβγ signaling coupled to c-Src activation of the Ras/MEK/ERK1/2 cascade. We show that, relative to normal cells, asthmatic HASM cells constitutively exhibit markedly increased intrinsic PDE4 activity coupled to heightened Gβγ-regulated phosphorylation of c-Src and ERK1/2, and direct co-localization of the latter with the PDE4D isoform. These signaling events and their induction of heightened PDE activity are acutely suppressed by treating asthmatic HASM cells with a Gβγ inhibitor. Importantly, along with increased Gβγ activation, asthmatic HASM cells also exhibit constitutively increased direct binding of the small Rap1 GTPase-activating protein, Rap1GAP, to the α-subunit of Gi protein, which serves to cooperatively facilitate Ras activation and, thereby, enable enhanced Gβγ-regulated ERK1/2-stimulated PDE activity. Collectively, these data are the first to identify that intrinsically increased signaling via the Gβγ subunit, facilitated by Rap1GAP recruitment to the α-subunit, mediates the constitutively increased PDE4 activity detected in asthmatic HASM cells. These new findings support the notion that interventions targeted at suppressing Gβγ signaling may lead to novel approaches to treat asthma.

EMMPRIN (CD147) Expression in Smooth Muscle Tumors of the Uterus.

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Kefeli, Mehmet; Yildiz, Levent; Gun, Seda; Ozen, Fatma Z; Karagoz, Filiz

2016-01-01

Smooth muscle tumors of the uterus are the most common mesenchymal tumors of the gynecologic tract. The vast majority of these are benign leiomyomas that present no diagnostic difficulty. Because some benign smooth muscle tumors may degenerate and uncommon variants exist, the diagnosis can be challenging in some cases. The goal of this research was to investigate EMMPRIN expression in leiomyomas, leiomyoma variants, and leiomyosarcomas (LMS) to determine whether it has a potential role in differential diagnosis. EMMPRIN expression was investigated with immunohistochemistry in 103 uterine smooth muscle tumors, which included 19 usual leiomyomas, 52 leiomyoma variants, and 32 LMS. They were evaluated on the basis of staining extent, intensity, and also their combined score, and the groups were compared. EMMPRIN expression was present in 3 of 19 (15.7%) usual leiomyomas, 23 of 52 (44.3%) leiomyoma variants, and 28 of 32 (87.5%) LMS. There were statistically significant differences in staining extent and intensity, and also for their combined scores, between the LMS and benign groups. Although uterine smooth muscle tumors are usually diagnosed easily with conventional diagnostic criteria, the differentiation of LMS from some variants of leiomyoma can be challenging based soley on morphology. EMMPRIN may be a valuable immunohistochemical marker for differentiating LMS from benign smooth muscle tumors in problematic cases.

Hypoxia Enhances Differentiation of Adipose Tissue-Derived Stem Cells toward the Smooth Muscle Phenotype

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Fang Wang

2018-02-01

Full Text Available Smooth muscle differentiated adipose tissue-derived stem cells are a valuable resource for regeneration of gastrointestinal tissues, such as the gut and sphincters. Hypoxia has been shown to promote adipose tissue-derived stem cells proliferation and maintenance of pluripotency, but the influence of hypoxia on their smooth myogenic differentiation remains unexplored. This study investigated the phenotype and contractility of adipose-derived stem cells differentiated toward the smooth myogenic lineage under hypoxic conditions. Oxygen concentrations of 2%, 5%, 10%, and 20% were used during differentiation of adipose tissue-derived stem cells. Real time reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of smooth muscle cells-specific markers, including early marker smooth muscle alpha actin, middle markers calponin, caldesmon, and late marker smooth muscle myosin heavy chain. The specific contractile properties of cells were verified with both a single cell contraction assay and a gel contraction assay. Five percent oxygen concentration significantly increased the expression levels of α-smooth muscle actin, calponin, and myosin heavy chain in adipose-derived stem cell cultures after 2 weeks of induction (p < 0.01. Cells differentiated in 5% oxygen conditions showed greater contraction effect (p < 0.01. Hypoxia influences differentiation of smooth muscle cells from adipose stem cells and 5% oxygen was the optimal condition to generate smooth muscle cells that contract from adipose stem cells.

The small GTPase Rac1 is required for smooth muscle contraction

DEFF Research Database (Denmark)

Rahman, Awahan; Davis, Benjamin; Lövdahl, Cecilia

2014-01-01

The role of the small GTP-binding protein Rac1 in smooth muscle contraction was examined using small molecule inhibitors (EHT1864, NSC23766) and a novel smooth muscle-specific, conditional, Rac1 knockout mouse strain. EHT1864, which affects nucleotide binding and inhibits Rac1 activity, concentra...

Evidence Supports Tradition: The in Vitro Effects of Roman Chamomile on Smooth Muscles

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Zsolt Sándor

2018-04-01

Full Text Available The dried flowers of Chamaemelum nobile (L. All. have been used in traditional medicine for different conditions related to the spasm of the gastrointestinal system. However, there have been no experimental studies to support the smooth muscle relaxant effect of this plant. The aim of our research was to assess the effects of the hydroethanolic extract of Roman chamomile, its fractions, four of its flavonoids (apigenin, luteolin, hispidulin, and eupafolin, and its essential oil on smooth muscles. The phytochemical compositions of the extract and its fractions were characterized and quantified by HPLC-DAD, the essential oil was characterized by GC and GC-MS. Neuronally mediated and smooth muscle effects were tested in isolated organ bath experiments on guinea pig, rat, and human smooth muscle preparations. The crude herbal extract induced an immediate, moderate, and transient contraction of guinea pig ileum via the activation of cholinergic neurons of the gut wall. Purinoceptor and serotonin receptor antagonists did not influence this effect. The more sustained relaxant effect of the extract, measured after pre-contraction of the preparations, was remarkable and was not affected by an adrenergic beta receptor antagonist. The smooth muscle-relaxant activity was found to be associated with the flavonoid content of the fractions. The essential oil showed only the relaxant effect, but no contracting activity. The smooth muscle-relaxant effect was also detected on rat gastrointestinal tissues, as well as on strip preparations of human small intestine. These results suggest that Roman chamomile extract has a direct and prolonged smooth muscle-relaxant effect on guinea pig ileum which is related to its flavonoid content. In some preparations, a transient stimulation of enteric cholinergic motoneurons was also detected. The essential oil also had a remarkable smooth muscle relaxant effect in this setting. Similar relaxant effects were also detected on

Evidence Supports Tradition: The in Vitro Effects of Roman Chamomile on Smooth Muscles.

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Sándor, Zsolt; Mottaghipisheh, Javad; Veres, Katalin; Hohmann, Judit; Bencsik, Tímea; Horváth, Attila; Kelemen, Dezső; Papp, Róbert; Barthó, Loránd; Csupor, Dezső

2018-01-01

The dried flowers of Chamaemelum nobile (L.) All. have been used in traditional medicine for different conditions related to the spasm of the gastrointestinal system. However, there have been no experimental studies to support the smooth muscle relaxant effect of this plant. The aim of our research was to assess the effects of the hydroethanolic extract of Roman chamomile, its fractions, four of its flavonoids (apigenin, luteolin, hispidulin, and eupafolin), and its essential oil on smooth muscles. The phytochemical compositions of the extract and its fractions were characterized and quantified by HPLC-DAD, the essential oil was characterized by GC and GC-MS. Neuronally mediated and smooth muscle effects were tested in isolated organ bath experiments on guinea pig, rat, and human smooth muscle preparations. The crude herbal extract induced an immediate, moderate, and transient contraction of guinea pig ileum via the activation of cholinergic neurons of the gut wall. Purinoceptor and serotonin receptor antagonists did not influence this effect. The more sustained relaxant effect of the extract, measured after pre-contraction of the preparations, was remarkable and was not affected by an adrenergic beta receptor antagonist. The smooth muscle-relaxant activity was found to be associated with the flavonoid content of the fractions. The essential oil showed only the relaxant effect, but no contracting activity. The smooth muscle-relaxant effect was also detected on rat gastrointestinal tissues, as well as on strip preparations of human small intestine. These results suggest that Roman chamomile extract has a direct and prolonged smooth muscle-relaxant effect on guinea pig ileum which is related to its flavonoid content. In some preparations, a transient stimulation of enteric cholinergic motoneurons was also detected. The essential oil also had a remarkable smooth muscle relaxant effect in this setting. Similar relaxant effects were also detected on other visceral

Identification and characterization of novel smoothelin isoforms in vascular smooth muscle.

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Krämer, J; Quensel, C; Meding, J; Cardoso, M C; Leonhardt, H

2001-01-01

Smoothelin is a cytoskeletal protein specifically expressed in differentiated smooth muscle cells and has been shown to colocalize with smooth muscle alpha actin. In addition to the small smoothelin isoform of 59 kD, we recently identified a large smoothelin isoform of 117 kD. The aim of this study was to identify and characterize novel smoothelin isoforms. The genomic structure and sequence of the smoothelin gene were determined by genomic PCR, RT-PCR and DNA sequencing. Comparison of the cDNA and genomic sequences shows that the small smoothelin isoform is generated by transcription initiation 10 kb downstream of the start site of the large isoform. In addition to the known smoothelin cDNA (c1 isoform) we identified two novel cDNA variants (c2 and c3 isoform) that are generated by alternative splicing within a region, which shows similarity to the spectrin family of F-actin cross-linking proteins. Visceral organs express the c1 form, while the c2 form prevails in well-vascularized tissue as analyzed by RT-PCR. We then generated specific antibodies against the major smoothelin isoforms and could show by Western blotting and immunohistochemistry that the large isoform is specifically expressed in vascular smooth muscle cells, while the small isoform is abundant in visceral smooth muscle. These results strongly suggest that the smoothelin gene contains a vascular and a visceral smooth muscle promoter. The cell-type-specific expression of smoothelin isoforms that are associated with actin filaments may play a role in the modulation of the contractile properties of different smooth muscle cell types. Copyright 2001 S. Karger AG, Basel

CXCL1 is a negative regulator of mast cell chemotaxis to airway smooth muscle cell products in vitro.

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Alkhouri, H; Moir, L M; Armour, C L; Hughes, J M

2014-03-01

Activated mast cells (MC) numbers on airway smooth muscle (ASM) are increased in eosinophilic asthma. In vitro, asthmatic cytokine-stimulated ASM cell-conditioned medium (CM) induces more MC chemotaxis than CM from nonasthmatic ASM cells. Intriguingly the nonasthmatic ASM CM inhibits MC chemotaxis to the asthmatic ASM CM. However, the inhibitory factor(s) in the nonasthmatic ASM CM is still to be identified. To identify the factor(s) released by nonasthmatic ASM cells that inhibits MC chemotaxis. Confluent, serum-starved ASM cells from donors with and without asthma were stimulated with IL-1β and T-helper (Th)1 (TNFα and IFNγ) or Th2 (IL-4, IL-13) cytokines, or left unstimulated. CM samples were collected after 24 h, and a potential inhibitory factor identified using cytokine protein arrays. Its production was assessed using ELISA and RT-PCR and inhibitory role investigated in MC chemotaxis and Ca(2+) mobilization assays. Only CXCL1 was produced in greater amounts by nonasthmatic than asthmatic ASM cells following Th1 and Th2 cytokine stimulation. CXCL1 mRNA expression was also increased. Exogenous rh-CXCL1 significantly inhibited MC intracellular Ca(2+) mobilization and chemotaxis to either CXCL10, CXCL8 or CM collected from asthmatic ASM cells following Th1 or Th2 cytokine stimulation. Neutralizing CXCL1 in nonasthmatic ASM CM or blocking its receptor significantly promoted MC chemotaxis. CXCL1 was a major factor regulating MC chemotaxis in vitro. Its differential release by ASM cells may explain the differences observed in MC localization to the ASM of people with and without asthma. CXCL1 inhibition of MC recruitment to the ASM may lead to new targets to limit asthma pathophysiology. © 2013 John Wiley & Sons Ltd.

Eye features in three Danish patients with multisystemic smooth muscle dysfunction syndrome

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Moller, Hans Ulrik; Fledelius, Hans C; Milewicz, Dianna M

2012-01-01

A de novo mutation of the ACTA2 gene encoding the smooth muscle cell α-actin has been established in patients with multisystemic smooth muscle dysfunction syndrome associated with patent ductus arteriosus and mydriasis present at birth....

Nitric Oxide Synthase Enzymes in the Airways of Mice Exposed to Ovalbumin: NOS2 Expression Is NOS3 Dependent

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Jennifer M. Bratt

2010-01-01

Full Text Available Objectives and Design. The function of the airway nitric oxide synthase (NOS isoforms and the lung cell types responsible for its production are not fully understood. We hypothesized that NO homeostasis in the airway is important to control inflammation, which requires upregulation, of NOS2 protein expression by an NOS3-dependent mechanism. Materials or Subjects. Mice from a C57BL/6 wild-type, NOS1−/−, NOS2−/−, and NOS3−/− genotypes were used. All mice strains were systemically sensitized and exposed to filtered air or ovalbumin (OVA aerosol for two weeks to create a subchronic model of allergen-induced airway inflammation. Methods. We measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled NO, nitrate and nitrite concentration, and airway NOS1, NOS2, and NOS3 protein content. Results. Deletion of NOS1 or NOS3 increases NOS2 protein present in the airway epithelium and smooth muscle of air-exposed animals. Exposure to allergen significantly reduced the expression of NOS2 protein in the airway epithelium and smooth muscle of the NOS3−/− strain only. This reduction in NOS2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express NOS2. NOS1−/− animals had significantly reduced goblet cell metaplasia compared to C57Bl/6 wt, NOS2−/−, and NOS3−/− allergen-exposed mice. Conclusion. The airway epithelial and smooth muscle cells maintain a stable airway NO concentration under noninflammatory conditions. This “homeostatic” mechanism is unable to distinguish between NOS derived from the different constitutive NOS isoforms. NOS3 is essential for the expression of NOS2 under inflammatory conditions, while NOS1 expression contributes to allergen-induced goblet cell metaplasia.

Nitric Oxide Synthase Enzymes in the Airways of Mice Exposed to Ovalbumin: NOS2 Expression Is NOS3 Dependent

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Bratt, Jennifer M.; Williams, Keisha; Rabowsky, Michelle F.; Last, Michael S.; Franzi, Lisa M.; Last, Jerold A.; Kenyon, Nicholas J.

2010-01-01

Objectives and Design. The function of the airway nitric oxide synthase (NOS) isoforms and the lung cell types responsible for its production are not fully understood. We hypothesized that NO homeostasis in the airway is important to control inflammation, which requires upregulation, of NOS2 protein expression by an NOS3-dependent mechanism. Materials or Subjects. Mice from a C57BL/6 wild-type, NOS1−/−, NOS2−/−, and NOS3−/− genotypes were used. All mice strains were systemically sensitized and exposed to filtered air or ovalbumin (OVA) aerosol for two weeks to create a subchronic model of allergen-induced airway inflammation. Methods. We measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled NO, nitrate and nitrite concentration, and airway NOS1, NOS2, and NOS3 protein content. Results. Deletion of NOS1 or NOS3 increases NOS2 protein present in the airway epithelium and smooth muscle of air-exposed animals. Exposure to allergen significantly reduced the expression of NOS2 protein in the airway epithelium and smooth muscle of the NOS3−/− strain only. This reduction in NOS2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express NOS2. NOS1−/− animals had significantly reduced goblet cell metaplasia compared to C57Bl/6 wt, NOS2−/−, and NOS3−/− allergen-exposed mice. Conclusion. The airway epithelial and smooth muscle cells maintain a stable airway NO concentration under noninflammatory conditions. This “homeostatic” mechanism is unable to distinguish between NOS derived from the different constitutive NOS isoforms. NOS3 is essential for the expression of NOS2 under inflammatory conditions, while NOS1 expression contributes to allergen-induced goblet cell metaplasia. PMID:20953358

From depolarization-dependent contractions in gastrointestinal smooth muscle to aortic pulse-synchronized contractions

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Marion SB

2014-03-01

Full Text Available Sarah B Marion, Allen W MangelRTI Health Solutions, Research Triangle Park, NC, USAAbstract: For decades, it was believed that the diameter of gastrointestinal smooth muscle cells is sufficiently narrow, and that the diffusion of calcium across the plasma membrane is sufficient, to support contractile activity. Thus, depolarization-triggered release of intracellular calcium was not believed to be operative in gastrointestinal smooth muscle. However, after the incubation of muscle segments in solutions devoid of calcium and containing the calcium chelator ethylene glycol tetraacetic acid, an alternative electrical event occurred that was distinct from normal slow waves and spikes. Subsequently, it was demonstrated in gastrointestinal smooth muscle segments that membrane depolarization associated with this alternative electrical event triggered rhythmic contractions by release of intracellular calcium. Although this concept of depolarization-triggered calcium release was iconoclastic, it has now been demonstrated in multiple gastrointestinal smooth muscle preparations. On the basis of these observations, we investigated whether a rhythmic electrical and mechanical event would occur in aortic smooth muscle under the same calcium-free conditions. The incubation of aortic segments in a solution with no added calcium plus ethylene glycol tetraacetic acid induced a fast electrical event without corresponding tension changes. On the basis of the frequency of these fast electrical events, we pursued, contrary to what has been established dogma for more than three centuries, the question of whether the smooth muscle wall of the aorta undergoes rhythmic activation during the cardiac cycle. As with depolarization-triggered contractile activity in gastrointestinal smooth muscle, it was “well known” that rhythmic activation of the aorta does not occur in synchrony with the heartbeat. In a series of experiments, however, it was demonstrated that rhythmic

Dietary Compound Kaempferol Inhibits Airway Thickening Induced by Allergic Reaction in a Bovine Serum Albumin-Induced Model of Asthma.

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Shin, Daekeun; Park, Sin-Hye; Choi, Yean-Jung; Kim, Yun-Ho; Antika, Lucia Dwi; Habibah, Nurina Umy; Kang, Min-Kyung; Kang, Young-Hee

2015-12-16

Asthma is characterized by aberrant airways including epithelial thickening, goblet cell hyperplasia, and smooth muscle hypertrophy within the airway wall. The current study examined whether kaempferol inhibited mast cell degranulation and prostaglandin (PG) release leading to the development of aberrant airways, using an in vitro model of dinitrophenylated bovine serum albumin (DNP-BSA)-sensitized rat basophilic leukemia (RBL-2H3) mast cells and an in vivo model of BSA-challenged asthmatic mice. Nontoxic kaempferol at 10-20 μM suppressed β-hexosaminidase release and cyclooxygenase 2 (COX2)-mediated production of prostaglandin D2 (PGD2) and prostaglandin F2α (PGF2α) in sensitized mast cells. Oral administration of ≤20 mg/kg kaempferol blocked bovine serum albumin (BSA) inhalation-induced epithelial cell excrescence and smooth muscle hypertrophy by attenuating the induction of COX2 and the formation of PGD2 and PGF2α, together with reducing the anti-α-smooth muscle actin (α-SMA) expression in mouse airways. Kaempferol deterred the antigen-induced mast cell activation of cytosolic phospholipase A2 (cPLA2) responsive to protein kinase Cμ (PKCμ) and extracellular signal-regulated kinase (ERK). Furthermore, the antigen-challenged activation of Syk-phospholipase Cγ (PLCγ) pathway was dampened in kaempferol-supplemented mast cells. These results demonstrated that kaempferol inhibited airway wall thickening through disturbing Syk-PLCγ signaling and PKCμ-ERK-cPLA2-COX2 signaling in antigen-exposed mast cells. Thus, kaempferol may be a potent anti-allergic compound targeting allergic asthma typical of airway hyperplasia and hypertrophy.

Characterising the mechanism of airway smooth muscle β2 adrenoceptor desensitization by rhinovirus infected bronchial epithelial cells.

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David Van Ly

Full Text Available Rhinovirus (RV infections account for approximately two thirds of all virus-induced asthma exacerbations and often result in an impaired response to β2 agonist therapy. Using an in vitro model of RV infection, we investigated the mechanisms underlying RV-induced β2 adrenoceptor desensitization in primary human airway smooth muscle cells (ASMC. RV infection of primary human bronchial epithelial cells (HBEC for 24 hours produced conditioned medium that caused β2 adrenoceptor desensitization on ASMCs without an effect on ASMCs viability. Less than 3 kDa size fractionation together with trypsin digestion of RV-induced conditioned medium did not prevent β2 adrenoceptor desensitization, suggesting it could potentially be mediated by a small peptide or lipid. RV infection of BECs, ASMCs and fibroblasts produced prostaglandins, of which PGE2, PGF2α and PGI2 had the ability to cause β2 adrenoceptor desensitization on ASMCs. RV-induced conditioned medium from HBECs depleted of PGE2 did not prevent ASMC β2 adrenoceptor desensitization; however this medium induced PGE2 from ASMCs, suggesting that autocrine prostaglandin production may be responsible. Using inhibitors of cyclooxygenase and prostaglandin receptor antagonists, we found that β2 adrenoceptor desensitization was mediated through ASMC derived COX-2 induced prostaglandins. Since ASMC prostaglandin production is unlikely to be caused by RV-induced epithelial derived proteins or lipids we next investigated activation of toll-like receptors (TLR by viral RNA. The combination of TLR agonists poly I:C and imiquimod induced PGE2 and β2 adrenoceptor desensitization on ASMC as did the RNA extracted from RV-induced conditioned medium. Viral RNA but not epithelial RNA caused β2 adrenoceptor desensitization confirming that viral RNA and not endogenous human RNA was responsible. It was deduced that the mechanism by which β2 adrenoceptor desensitization occurs was by pattern recognition receptor

Fragmented esophageal smooth muscle contraction segments on high resolution manometry: a marker of esophageal hypomotility.

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Porter, R F; Kumar, N; Drapekin, J E; Gyawali, C P

2012-08-01

Esophageal peristalsis consists of a chain of contracting striated and smooth muscle segments on high resolution manometry (HRM). We compared smooth muscle contraction segments in symptomatic subjects with reflux disease to healthy controls. High resolution manometry Clouse plots were analyzed in 110 subjects with reflux disease (50 ± 1.4 years, 51.5% women) and 15 controls (27 ± 2.1 years, 60.0% women). Using the 30 mmHg isobaric contour tool, sequences were designated fragmented if either smooth muscle contraction segment was absent or if the two smooth muscle segments were separated by a pressure trough, and failed if both smooth muscle contraction segments were absent. The discriminative value of contraction segment analysis was assessed. A total of 1115 swallows were analyzed (reflux group: 965, controls: 150). Reflux subjects had lower peak and averaged contraction amplitudes compared with controls (P value to HRM analysis. Specifically, fragmented smooth muscle contraction segments may be a marker of esophageal hypomotility. © 2012 Blackwell Publishing Ltd.

Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

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Brun, Juliane; Lutz, Katrin A; Neumayer, Katharina M H; Klein, Gerd; Seeger, Tanja; Uynuk-Ool, Tatiana; Wörgötter, Katharina; Schmid, Sandra; Kraushaar, Udo; Guenther, Elke; Rolauffs, Bernd; Aicher, Wilhelm K; Hart, Melanie L

2015-01-01

The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel

Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

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Juliane Brun

Full Text Available The use of mesenchymal stromal cells (MSCs differentiated toward a smooth muscle cell (SMC phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2, transgelin (TAGLN, calponin (CNN1, and smooth muscle myosin heavy chain (SM-MHC; MYH11 according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion

Regulation of collagen biosynthesis in cultured bovine aortic smooth muscle cells

International Nuclear Information System (INIS)

Stepp, M.A.

1986-01-01

Aortic smooth muscles cells have been implicated in the etiology of lesions which occur in atherosclerosis and hypertension. Both diseases involve proliferation of smooth muscle cells and accumulation of excessive amounts of extracellular matrix proteins, including collagen type I and type III produced by the smooth muscle cells. To better understand the sites of regulation of collagen biosynthesis and to correlate these with the growth rate of the cells, cultured bovine aortic smooth muscle cells were studied as a function of the number of days (3 to 14) in second passage. Cells grew rapidly up to day 6 when confluence was reached. The total incorporation of [ 3 H]-proline into proteins was highest at day 3 and decreased to a constant level after the cultures reached confluence. In contrast, collagen protein production was lowest before confluence and continued to increase over the entire time course of the experiments. cDNA clones for the α1 and α2 chains of type I and the α1 chain of type III collagen were used to quantitate the steady state level of collagen mRNAs. RNA was tested in a cell-free translation system. Changes in the translational activity of collagen mRNAs parallelled the observed increases in collagen protein production. Thus, at later time points, collagen mRNAs are more active in directing synthesis of preprocollagens, even though less collagen mRNA is present. The conclusion is that the site of regulation of the expression of collagen genes is a function of the growth rate of cultured smooth muscle cells

Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.

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Liu, Dong-Hai; Huang, Xu; Guo, Xin; Meng, Xiang-Min; Wu, Yi-Song; Lu, Hong-Li; Zhang, Chun-Mei; Kim, Young-chul; Xu, Wen-Xie

2014-01-01

Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV) was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV) to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.

Patent ductus arteriosus in mice with smooth muscle-specific Jag1 deletion

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Feng, Xuesong; Krebs, Luke T.; Gridley, Thomas

2010-01-01

The ductus arteriosus is an arterial vessel that shunts blood flow away from the lungs during fetal life, but normally occludes after birth to establish the adult circulation pattern. Failure of the ductus arteriosus to close after birth is termed patent ductus arteriosus and is one of the most common congenital heart defects. Mice with smooth muscle cell-specific deletion of Jag1, which encodes a Notch ligand, die postnatally from patent ductus arteriosus. These mice exhibit defects in contractile smooth muscle cell differentiation in the vascular wall of the ductus arteriosus and adjacent descending aorta. These defects arise through an inability to propagate the JAG1-Notch signal via lateral induction throughout the width of the vascular wall. Both heterotypic endothelial smooth muscle cell interactions and homotypic vascular smooth muscle cell interactions are required for normal patterning and differentiation of the ductus arteriosus and adjacent descending aorta. This new model for a common congenital heart defect provides novel insights into the genetic programs that underlie ductus arteriosus development and closure. PMID:21068062

Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.

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Kim, Kyoungtae; Keller, Thomas C S

2002-01-07

Smooth muscle cells use an actin-myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

Notch signal reception is required in vascular smooth muscle cells for ductus arteriosus closure

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Krebs, Luke T.; Norton, Christine R.; Gridley, Thomas

2017-01-01

Summary The ductus arteriosus is an arterial vessel that shunts blood flow away from the lungs during fetal life, but normally occludes after birth to establish the adult circulation pattern. Failure of the ductus arteriosus to close after birth is termed patent ductus arteriosus, and is one of the most common congenital heart defects. Our previous work demonstrated that vascular smooth muscle cell expression of the Jag1 gene, which encodes a ligand for Notch family receptors, is essential for postnatal closure of the ductus arteriosus in mice. However, it was not known what cell population was responsible for receiving the Jag1-mediated signal. Here we show, using smooth muscle cell-specific deletion of the Rbpj gene, which encodes a transcription factor that mediates all canonical Notch signaling, that Notch signal reception in the vascular smooth muscle cell compartment is required for ductus arteriosus closure. These data indicate that homotypic vascular smooth muscle cell interactions are required for proper contractile smooth muscle cell differentiation and postnatal closure of the ductus arteriosus in mice. PMID:26742650

Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.

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Dong-Hai Liu

Full Text Available Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.

Rac1 modulates G-protein-coupled receptor-induced bronchial smooth muscle contraction.

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Sakai, Hiroyasu; Kai, Yuki; Sato, Ken; Ikebe, Mitsuo; Chiba, Yohihiko

2018-01-05

Increasing evidence suggests a functional role of RhoA/Rho-kinase signalling as a mechanism for smooth muscle contraction; however, little is known regarding the roles of Rac1 and other members of the Rho protein family. This study aimed to examine whether Rac1 modulates bronchial smooth muscle contraction. Ring preparations of bronchi isolated from rats were suspended in an organ bath, and isometric contraction of circular smooth muscle was measured. Immunoblotting was used to examine myosin light chain phosphorylation in bronchial smooth muscle. Our results demonstrated that muscle contractions induced by carbachol (CCh) and endothelin-1 (ET-1) were inhibited by EHT1864, a selective Rac1 inhibitor, and NSC23766, a selective inhibitor of Rac1-specific guanine nucleotide exchange factors. Similarly, myosin light chain and myosin phosphatase target subunit 1 (MYPT1) at Thr853 phosphorylation induced by contractile agonist were inhibited with Rac1 inhibition. However, contractions induced by high K + , calyculin A (a potent protein phosphatase inhibitor) and K + /PDBu were not inhibited by these Rac1 inhibitors. Interestingly, NaF (a G-protein activator)-induced contractions were inhibited by EHT1864 but not by NSC23766. We next examined the effects of a trans-acting activator of transcription protein transduction domain (PTD) fusion protein with Rac1 (PTD-Rac1) on muscle contraction. The constitutively active form of PTD-Rac1 directly induced force development and contractions were abolished by EHT1864. These results suggest that Rac1, activated by G protein-coupled receptor agonists, such as CCh and ET-1, may induce myosin light chain and MYPT phosphorylation and modulate the contraction of bronchial smooth muscle. Copyright © 2017 Elsevier B.V. All rights reserved.

TGF-β-activated kinase 1 (TAK1 signaling regulates TGF-β-induced WNT-5A expression in airway smooth muscle cells via Sp1 and β-catenin.

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Kuldeep Kumawat

Full Text Available WNT-5A, a key player in embryonic development and post-natal homeostasis, has been associated with a myriad of pathological conditions including malignant, fibroproliferative and inflammatory disorders. Previously, we have identified WNT-5A as a transcriptional target of TGF-β in airway smooth muscle cells and demonstrated its function as a mediator of airway remodeling. Here, we investigated the molecular mechanisms underlying TGF-β-induced WNT-5A expression. We show that TGF-β-activated kinase 1 (TAK1 is a critical mediator of WNT-5A expression as its pharmacological inhibition or siRNA-mediated silencing reduced TGF-β induction of WNT-5A. Furthermore, we show that TAK1 engages p38 and c-Jun N-terminal kinase (JNK signaling which redundantly participates in WNT-5A induction as only simultaneous, but not individual, inhibition of p38 and JNK suppressed TGF-β-induced WNT-5A expression. Remarkably, we demonstrate a central role of β-catenin in TGF-β-induced WNT-5A expression. Regulated by TAK1, β-catenin is required for WNT-5A induction as its silencing repressed WNT-5A expression whereas a constitutively active mutant augmented basal WNT-5A abundance. Furthermore, we identify Sp1 as the transcription factor for WNT-5A and demonstrate its interaction with β-catenin. We discover that Sp1 is recruited to the WNT-5A promoter in a TGF-β-induced and TAK1-regulated manner. Collectively, our findings describe a TAK1-dependent, β-catenin- and Sp1-mediated signaling cascade activated downstream of TGF-β which regulates WNT-5A induction.

Airway reactivity in chronic obstructive pulmonary disease. Failure of in vivo methacholine responsiveness to correlate with cholinergic, adrenergic, or nonadrenergic responses in vitro.

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Taylor, S M; Paré, P D; Armour, C L; Hogg, J C; Schellenberg, R R

1985-07-01

This study aimed to determine whether in vivo airways hyperreactivity was manifested by either enhanced bronchial smooth muscle responses to contractile stimuli or by deficient responses to relaxant stimuli in vitro. Quantitative responses to nebulized methacholine were obtained in 12 human subjects prior to pulmonary resection. The provocative concentration of methacholine producing a 20% reduction in FEV1 (PC20) was calculated, and these values were compared with in vitro responses of bronchial smooth muscle strips from the surgical specimens. Both contractile cholinergic responses and relaxant nonadrenergic noncholinergic dose-response data were obtained for the in vitro bronchial specimens by electrical field stimulation. In addition, cumulative dose responses were obtained to exogenously added methacholine, the beta-adrenergic agonist salbutamol, and the adenylate cyclase activator forskolin. Despite a wide range of PC20 values, the in vivo airway responsiveness did not correlate with any of the in vitro responses examined, suggesting that airway reactivity is not due solely to the responsiveness of smooth muscle to contractile agonists nor to a localized deficiency in the nonadrenergic inhibitory system, beta-adrenergic inhibition, or abnormal cyclic-AMP-mediated pathways of relaxation.

Cordyceps sinensis inhibits airway remodeling in rats with chronic obstructive pulmonary disease.

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Yang, Lei; Jiao, Xingai; Wu, Jinxiang; Zhao, Jiping; Liu, Tian; Xu, Jianfeng; Ma, Xiaohui; Cao, Liuzao; Liu, Lin; Liu, Yahui; Chi, Jingyu; Zou, Minfang; Li, Shuo; Xu, Jiawei; Dong, Liang

2018-03-01

Cordyceps sinensis is a traditional Chinese herbal medicine that has been used for centuries in Asia as a tonic to soothe the lung for the treatment of respiratory diseases. The aim of the present study was to determine the effects of C. sinensi s on airway remodeling in chronic obstructive pulmonary disease (COPD) and investigate the underlying molecular mechanisms. Rats with COPD were orally administered C. sinensis at low, moderate or high doses (2.5, 5 or 7.5 g/kg/day, respectively) for 12 weeks. Airway tissue histopathology, lung inflammation and airway remodeling were evaluated. C. sinensis treatment significantly ameliorated airway wall thickening, involving collagen deposition, airway wall fibrosis, smooth muscle hypertrophy and epithelial hyperplasia in model rats with COPD. Additionally, C. sinensis administration in rats with COPD reduced inflammatory cell accumulation and decreased inflammatory cytokine production, including tumor necrosis factor-α, interleukin-8 and transforming growth factor (TGF)-β1 in bronchoalveolar lavage fluid. Meanwhile, the increased levels of α-smooth muscle actin and collagen I in the COPD group were also markedly decreased by C. sinensis treatment. Furthermore, compared with untreated rats with COPD, C. sinensis reduced the expression level of phosphorylated (p)-Smad2, p-Smad3, TGF-β1 and its receptors, with the concomitant increased expression of Smad7 in the lungs of rats with COPD. These results indicated that treatment with C. sinensis may be a useful approach for COPD therapy.

Pathologic bladder microenvironment attenuates smooth muscle differentiation of skin derived precursor cells: implications for tissue regeneration.

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Cornelia Tolg

Full Text Available Smooth muscle cell containing organs (bladder, heart, blood vessels are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.

Effects of nifedipine on anorectal smooth muscle in vitro.

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Cook, T A; Brading, A F; Mortensen, N J

1999-06-01

Glyceryl trinitrate reduces anal resting pressure and aids the healing of anal fissures. However, some patients develop tachyphylaxis and the fissure fails to heal, suggesting that other agents are needed. This study assesses the effects of nifedipine (a calcium channel antagonist) in modulating resting tone and agonist-induced contractions in human internal anal sphincter (IAS) and rectal circular muscle. Smooth muscle strips from the IAS and rectal circular muscle from ten patients undergoing surgical resection were mounted for isometric tension recording in a superfusion organ bath. The effects of noradrenaline and carbachol were assessed in the presence of various perfusates. LAS strips developed tone and spontaneous activity. Noradrenaline produced dose-dependent contractions. In calcium-free Krebs solution, tone and activity were abolished and no contractions were elicited in response to noradrenaline. Nifedipine also abolished tone and spontaneous activity, but contractions to noradrenaline were only slightly attenuated. In contrast, rectal smooth muscle strips developed spontaneous activity but no resting tone and contracted in response to carbachol. In calcium-free Krebs solution, the spontaneous activity and carbachol contractions were abolished. Addition of nifedipine to the perfusate abolished spontaneous activity and greatly reduced contractions. These data suggest that spontaneous activity and resting tone are dependent on extracellular calcium and flux across the cells. Agonist-induced contraction in the IAS is attributable mainly to the release of calcium from intracellular stores, whereas rectal circular smooth muscle depends principally on extracellular calcium entering the cell for contraction. The attenuation of contractions in both tissues and the abolition of resting tone in the IAS suggest that nifedipine may be useful in the management of patients with anorectal disorders.

Expression profile and protein translation of TMEM16A in murine smooth muscle

DEFF Research Database (Denmark)

Davis, Alison J; Forrest, Abigail S; Jepps, Thomas Andrew

2010-01-01

Recently, overexpression of the genes TMEM16A and TMEM16B has been shown to produce currents qualitatively similar to native Ca(2+)-activated Cl(-) currents (I(ClCa)) in vascular smooth muscle. However, there is no information about this new gene family in vascular smooth muscle, where Cl(-) chan...

Electron histochemical and autoradiographic studies of vascular smooth muscle cell

International Nuclear Information System (INIS)

Kameyama, Kohji; Aida, Takeo; Asano, Goro

1982-01-01

The authors have studied the vascular smooth muscle cell in the aorta and the arteries of brain, heart in autopsied cases, cholesterol fed rabbits and canine through electron histochemical and autoradiographic methods, using 3 H-proline and 3 H-thymidine. The vascular changes are variable presumably due to the functional and morphological difference of vessels. Aging, pathological condition and physiological requirement induce the disturbances of vascular functions as contractility. According to various pathological conditions, the smooth muscle cell altered their shape, surface properties and arrangement of subcellular organelles including changes in number. The morphological features of arteries during aging is characterized by the thickening of endothelium and media. Decreasing cellularity and increasing collagen contents in media. The autoradiographic and histochemical observations using periodic acid methenamine silver (PAM) and ruthenium red stains demonstrated that the smooth muscle cell is a connective tissue synthetic cell. The PAM impregnation have proved that the small bundle of microfilaments become associated with small conglomerate of collagen and elastic fibers. Cytochemical examination will provide sufficient evidence to establish the contribution of subcellular structure. The acid phosphatase play an important role in vascular disease and they are directly involved in cellular lipid metabolism in cholesterol fed animals, and the activity of Na-K ATPase on the plasma membrane may contribute to the regulation of vascular blood flow and vasospasms. Direct injury and subsequent abnormal contraction of smooth muscle cell may initiate increased permeability of plasma protein and lipid in the media layer and eventually may developed and enhance arteriosclerosis. (author)

L-Thyroxine promotes a proliferative airway smooth muscle phenotype in the presence of TGF-β1

NARCIS (Netherlands)

Dekkers, Bart G J; Naeimi, Saeideh; Bos, I. Sophie T.; Menzen, Mark H; Halayko, Andrew John; Sadeghi Hashjin, Goudarz; Meurs, Herman

2015-01-01

Hypothyroidism may reduce, whereas hyperthyroidism may aggravate asthma symptoms. The mechanisms underlying this relationship are largely unknown. Since thyroid hormones have central roles in cell growth and differentiation, we hypothesized that airway remodeling, in particular increased airway

Effects of age on muscarinic agonist-induced contraction an IP accumulation in airway smooth muscle

International Nuclear Information System (INIS)

Wills-Karp, M.

1991-01-01

The effects of age on carbachol-stimulated force development and [ 3 H]inositol phosphate production was studied in tracheal rings from guinea pigs aged 1 month and 25 months of age. The pD 2 for the contractile response to carbachol was significantly reduced in tracheal tissues from old animals as compared to that of the young tissues, respectively. In contrast, inositol phosphate formation was not altered with increasing age when stimulated by carbachol or NaF, a direct activator of G proteins. Carbachol-induced inositol phosphate accumulation was inhibited by treatment with 1μg/ml pertussis toxin, suggesting that IP1 accumulation is coupled to a pertussis-toxin-sensitive protein. The pD 2 values for contraction were significantly different from the pD 2 values for IP1 accumulation, in both young and old tissues, respectively. These data suggest that IP1 accumulation is not responsible for the decreased contractile ability in tracheal smooth muscle during aging

Effects of age on muscarinic agonist-induced contraction an IP accumulation in airway smooth muscle

Energy Technology Data Exchange (ETDEWEB)

Wills-Karp, M. (Johns Hopkins Univ., Baltimore, MD (United States))

1991-01-01

The effects of age on carbachol-stimulated force development and ({sup 3}H)inositol phosphate production was studied in tracheal rings from guinea pigs aged 1 month and 25 months of age. The pD{sub 2} for the contractile response to carbachol was significantly reduced in tracheal tissues from old animals as compared to that of the young tissues, respectively. In contrast, inositol phosphate formation was not altered with increasing age when stimulated by carbachol or NaF, a direct activator of G proteins. Carbachol-induced inositol phosphate accumulation was inhibited by treatment with 1{mu}g/ml pertussis toxin, suggesting that IP1 accumulation is coupled to a pertussis-toxin-sensitive protein. The pD{sub 2} values for contraction were significantly different from the pD{sub 2} values for IP1 accumulation, in both young and old tissues, respectively. These data suggest that IP1 accumulation is not responsible for the decreased contractile ability in tracheal smooth muscle during aging.

on Isolated Smooth Muscle Preparation in Rats

African Journals Online (AJOL)

Samuel Olaleye

ABSTRACT. This study investigated the receptor effects of methanolic root extract of ... Phytochemical Analysis: Photochemistry of the methanolic extract was ... mounted with resting tension 0.5g in an organ bath containing .... Effects of extra cellular free Ca2+ and 0.5mM ... isolated smooth muscle by high K+ on the other.

Coordinated Respiratory Motor Activity in Nerves Innervating the Upper Airway Muscles in Rats.

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Satoshi Tachikawa

Full Text Available Maintaining the patency of the upper airway during breathing is of vital importance. The activity of various muscles is related to the patency of the upper airway. In the present study, we examined the respiratory motor activity in the efferent nerves innervating the upper airway muscles to determine the movements of the upper airway during respiration under normocapnic conditions (pH = 7.4 and in hypercapnic acidosis (pH = 7.2. Experiments were performed on arterially perfused decerebrate rats aged between postnatal days 21-35. We recorded the efferent nerve activity in a branch of the cervical spinal nerve innervating the infrahyoid muscles (CN, the hypoglossal nerve (HGN, the external branch of the superior laryngeal nerve (SLN, and the recurrent laryngeal nerve (RLN with the phrenic nerve (PN. Inspiratory nerve discharges were observed in all these nerves under normocapnic conditions. The onset of inspiratory discharges in the CN and HGN was slightly prior to those in the SLN and RLN. When the CO2 concentration in the perfusate was increased from 5% to 8% to prepare for hypercapnic acidosis, the peak amplitudes of the inspiratory discharges in all the recorded nerves were increased. Moreover, hypercapnic acidosis induced pre-inspiratory discharges in the CN, HGN, SLN, and RLN. The onset of pre-inspiratory discharges in the CN, HGN, and SLN was prior to that of discharges in the RLN. These results suggest that the securing of the airway that occurs a certain time before dilation of the glottis may facilitate ventilation and improve hypercapnic acidosis.

Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action.

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Fan, Ya-Ping; Puri, Rajinder N; Rattan, Satish

2002-03-01

Effect of ANG II was investigated in in vitro smooth muscle strips and in isolated smooth muscle cells (SMC). Among different species, rat internal and sphincter (IAS) smooth muscle showed significant and reproducible contraction that remained unmodified by different neurohumoral inhibitors. The AT(1) antagonist losartan but not AT(2) antagonist PD-123319 antagonized ANG II-induced contraction of the IAS smooth muscle and SMC. ANG II-induced contraction of rat IAS smooth muscle and SMC was attenuated by tyrosine kinase inhibitors genistein and tyrphostin, protein kinase C (PKC) inhibitor H-7, Ca(2+) channel blocker nicardipine, Rho kinase inhibitor Y-27632 or p(44/42) mitogen-activating protein kinase (MAPK(44/42)) inhibitor PD-98059. Combinations of nicardipine and H-7, Y-27632, and PD-98059 caused further attenuation of the ANG II effects. Western blot analyses revealed the presence of both AT(1) and AT(2) receptors. We conclude that ANG II causes contraction of rat IAS smooth muscle by the activation of AT(1) receptors at the SMC and involves multiple intracellular pathways, influx of Ca(2+), and activation of PKC, Rho kinase, and MAPK(44/42).

Airway resistance at maximum inhalation as a marker of asthma and airway hyperresponsiveness

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O'Connor George T

2011-07-01

Full Text Available Abstract Background Asthmatics exhibit reduced airway dilation at maximal inspiration, likely due to structural differences in airway walls and/or functional differences in airway smooth muscle, factors that may also increase airway responsiveness to bronchoconstricting stimuli. The goal of this study was to test the hypothesis that the minimal airway resistance achievable during a maximal inspiration (Rmin is abnormally elevated in subjects with airway hyperresponsiveness. Methods The Rmin was measured in 34 nonasthmatic and 35 asthmatic subjects using forced oscillations at 8 Hz. Rmin and spirometric indices were measured before and after bronchodilation (albuterol and bronchoconstriction (methacholine. A preliminary study of 84 healthy subjects first established height dependence of baseline Rmin values. Results Asthmatics had a higher baseline Rmin % predicted than nonasthmatic subjects (134 ± 33 vs. 109 ± 19 % predicted, p = 0.0004. Sensitivity-specificity analysis using receiver operating characteristic curves indicated that baseline Rmin was able to identify subjects with airway hyperresponsiveness (PC20 min % predicted, FEV1 % predicted, and FEF25-75 % predicted, respectively. Also, 80% of the subjects with baseline Rmin min > 145% predicted had hyperresponsive airways, regardless of clinical classification as asthmatic or nonasthmatic. Conclusions These findings suggest that baseline Rmin, a measurement that is easier to perform than spirometry, performs as well as or better than standard spirometric indices in distinguishing subjects with airway hyperresponsiveness from those without hyperresponsive airways. The relationship of baseline Rmin to asthma and airway hyperresponsiveness likely reflects a causal relation between conditions that stiffen airway walls and hyperresponsiveness. In conjunction with symptom history, Rmin could provide a clinically useful tool for assessing asthma and monitoring response to treatment.

Upper airway muscles awake and asleep.

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Sériès, Frédéric

2002-06-01

Upper airway (UA) structures are involved in different respiratory and non-respiratory tasks. The coordination of agonist and antagonist UA dilators is responsible for their mechanical function and their ability to maintain UA patency throughout the respiratory cycle. The activity of these muscles is linked with central respiratory activity but also depends on UA pressure changes and is greatly influenced by sleep. UA muscles are involved in determining UA resistance and stability (i.e. closing pressure), and the effect of sleep on these variables may be accounted for by its effect on tonic and phasic skeletal muscle activities. The mechanical effects of UA dilator contraction also depend on their physiological properties (capacity to generate tension in vitro, activity of the anaerobic enzymatic pathway, histo-chemical characteristics that may differ between subjects who may or may not have sleep-related obstructive breathing disorders). These characteristics may represent an adaptive process to an increased resistive loading of these muscles. The apparent discrepancy between the occurrence of UA closure and an increased capacity to generate tension in sleep apnea patients may be due to a reduction in the effectiveness of UA muscle contraction in these patients; such an increase in tissue stiffness could be accounted for by peri-muscular tissue characteristics. Therefore, understanding of UA muscle physiological characteristics should take into account its capacity for force production and its mechanical coupling with other UA tissues. Important research goals for the future will be to integrate these issues with other physiological features of the disease, such as UA size and dimension, histological characteristics of UA tissues and the effect of sleep on muscle function. Such integration will better inform understanding of the role of pharyngeal UA muscles in the pathophysiology of the sleep apnea/hypopnea syndrome.

Effects of prostaglandin E2 and cAMP elevating drugs on GM-CSF release by cultured human airway smooth muscle cells. Relevance to asthma therapy.

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Lazzeri, N; Belvisi, M G; Patel, H J; Yacoub, M H; Chung, K F; Mitchell, J A

2001-01-01

Human airway smooth muscle (HASM) cells release granulocyte macrophage-colony stimulating factor (GM-CSF) and express cyclooxygenase (COX)-2 (resulting in the release of prostaglandin [PG] E2) after stimulation with cytokines. Because COX-2 activity can regulate a number of inflammatory processes, we have assessed its effects, as well as those of agents that modulate cyclic adenosine monophosphate (cAMP), on GM-CSF release by HASM cells. Cells stimulated with a combination of proinflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha each at 10 ng/ml) for 24 h released significant amounts of PGE2 (measured by radioimmunoassay) and GM-CSF (measured by enzyme-linked immunosorbent assay). Indomethacin and other COX-1/COX-2 inhibitors caused concentration-dependent inhibitions of PGE2 concomitantly with increases in GM-CSF formation. Addition of exogenous PGE2 or the beta2-agonist fenoterol, which increase cAMP, to cytokine-treated HASM cells had no effect on GM-CSF release unless COX activity was first blocked with indomethacin. The type 4 phosphodiesterase inhibitors rolipram and SB 207499 both caused concentration-dependent reductions in GM-CSF production. Thus, when HASM cells are activated with cytokines they release PGE2, which acts as a "braking mechanism" to limit the coproduction of GM-CSF. Moreover, agents that elevate cAMP also reduce GM-CSF formation by these cells.

The Smooth Muscle of the Artery

Science.gov (United States)

1975-01-01

of vascular smooth muscle are contrac- tion, thereby mediating vaso constriction, and the synthesis of the extracellular proteins and polysaccharides ...of the monosaccharides turned out to be different for instance from cornea to aorta (229, 283). In the conditions yed (4 hours incubation at 37 degrees... polysaccharides only. This glyco- protein is not very rich in sugar components (- 5Z) (228, 284), but is a very acidic protein (286). Fig.66 shows

Experimental model of human corpus cavernosum smooth muscle relaxation

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Rommel P. Regadas

2010-08-01

Full Text Available PURPOSE: To describe a technique for en bloc harvesting of the corpus cavernosum, cavernous artery and urethra from transplant organ donors and contraction-relaxation experiments with corpus cavernosum smooth muscle. MATERIALS AND METHODS: The corpus cavernosum was dissected to the point of attachment with the crus penis. A 3 cm segment (corpus cavernosum and urethra was isolated and placed in ice-cold sterile transportation buffer. Under magnification, the cavernous artery was dissected. Thus, 2 cm fragments of cavernous artery and corpus cavernosum were obtained. Strips measuring 3 x 3 x 8 mm3 were then mounted vertically in an isolated organ bath device. Contractions were measured isometrically with a Narco-Biosystems force displacement transducer (model F-60, Narco-Biosystems, Houston, TX, USA and recorded on a 4-channel Narco-Biosystems desk model polygraph. RESULTS: Phenylephrine (1µM was used to induce tonic contractions in the corpus cavernosum (3 - 5 g tension and cavernous artery (0.5 - 1g tension until reaching a plateau. After precontraction, smooth muscle relaxants were used to produce relaxation-response curves (10-12M to 10-4 M. Sodium nitroprusside was used as a relaxation control. CONCLUSION: The harvesting technique and the smooth muscle contraction-relaxation model described in this study were shown to be useful instruments in the search for new drugs for the treatment of human erectile dysfunction.

Cultured smooth muscle cells of the human vesical sphincter are more sensitive to histamine than are detrusor smooth muscle cells.

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Neuhaus, Jochen; Oberbach, Andreas; Schwalenberg, Thilo; Stolzenburg, Jens-Uwe

2006-05-01

To compare histamine receptor expression in cultured smooth muscle cells from the human detrusor and internal sphincter using receptor-specific agonists. Smooth muscle cells from the bladder dome and internal sphincter were cultured from 5 male patients undergoing cystectomy for bladder cancer therapy. Calcium transients in cells stimulated with carbachol, histamine, histamine receptor 1 (H1R)-specific heptanecarboxamide (HTMT), dimaprit (H2R), and R-(alpha)-methylhistamine (H3R) were measured by calcium imaging. Histamine receptor proteins were detected by Western blot analysis and immunocytochemistry. H1R, H2R, and H3R expression was found in tissue and cultured cells. Carbachol stimulated equal numbers of detrusor and sphincter cells (60% and 51%, respectively). Histamine stimulated significantly more cells than carbachol in detrusor (100%) and sphincter (99.34%) cells. Calcium responses to carbachol in detrusor and sphincter cells were comparable and did not differ from those to histamine in detrusor cells. However, histamine and specific agonists stimulated more sphincter cells than did carbachol (P <0.001), and the calcium increase was greater in sphincter cells than in detrusor cells. Single cell analysis revealed comparable H2R responses in detrusor and sphincter cells, but H1R and H3R-mediated calcium reactions were significantly greater in sphincter cells. Histamine very effectively induces calcium release in smooth muscle cells. In sphincter cells, histamine is even more effective than carbachol regarding the number of reacting cells and the intracellular calcium increase. Some of the variability in the outcome of antihistaminic interstitial cystitis therapies might be caused by the ineffectiveness of the chosen antihistaminic or unintentional weakening of sphincteric function.

Evidence of direct smooth muscle relaxant effects of the fibrate gemfibrozil.

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Phelps, Laura E; Peuler, Jacob D

2010-01-01

Fibrates are commonly employed to treat abnormal lipid metabolism via their unique ability to stimulate peroxisome proliferator-activated receptor alpha (PPARalpha). Interestingly, they also decrease systemic arterial pressure, despite recent evidence that PPAR alpha may contribute to expression of renin and related hypertension. Yet, mechanisms responsible for their potential antihypertensive activity remain unresolved. Rapid decreases in arterial pressure following bolus intravenous injections of bezafibrate strongly suggest they may relax arterial smooth muscle directly. But since bezafibrate is highly susceptible to photodegradation in aqueous media, it has never been critically tested for this possibility in vitro with isolated arterial smooth muscle preparations. Accordingly, we tested gemfibrozil which is resistant to photodegradation. We examined it over a therapeutically-relevant range (50-400 microM) for both acute and delayed relaxant effects on contractions of the isolated rat tail artery; contractions induced by either depolarizing its smooth muscle cell membranes with high potassium or stimulating its membrane-bound receptors with norepinephrine and arginine-vasopressin. We also examined these same gemfibrozil levels for effects on spontaneously-occurring phasic rhythmic contractile activity, typically not seen in arteries under in vitro conditions but commonly exhibited by smooth muscle of uterus, duodenum and bladder. We found that gemfibrozil significantly relaxed all induced forms of contraction in the rat tail artery, acutely at the higher test levels and after a delay of a few hours at the lower test levels. The highest test level of gemfibrozil (400 microM) also completely abolished spontaneously-occurring contractile activity of the isolated uterus and duodenum and markedly suppressed it in the bladder. This is the first evidence that a fibrate drug can directly relax smooth muscle contractions, either induced by various contractile agents or

Smooth muscle-like tissue constructs with circumferentially oriented cells formed by the cell fiber technology.

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Hsiao, Amy Y; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

2015-01-01

The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.

Effects of (-)-desmethoxyverapamil on heart and vascular smooth muscle

International Nuclear Information System (INIS)

Nawrath, H.; Raschack, M.

1987-01-01

(-)-Desmethoxyverapamil [also known as (-)-devapamil or (-)-D888] has been developed as a verapamil type radioligand for the study of calcium channels. In the present investigation, the effects of (-)-desmethoxyverapamil on action potential (AP) and force of contraction in heart muscle preparations and on tension and 45 Ca influx in vascular smooth muscle are described. In part, the effects were compared with the (+)-isomer of desmethoxyverapamil and the isomers of both verapamil and methoxyverapamil. In atrial and/or ventricular heart muscle preparations from guinea pigs, cats and man, (-)-desmethoxyverapamil decreased the force of contraction and shortened the AP duration. Slow response APs were depressed, whereas dV/dtmax of phase 0 of the AP remained unchanged. The rank order of potency of the (-)-isomers was as follows: desmethoxyverapamil greater than methoxyverapamil greater than verapamil. Potassium-induced contractures and 45 Ca influx were depressed by the (-)-isomers of desmethoxyverapamil, methoxyverapamil and verapamil in the same potency rank order as observed in heart muscle. The (+)-isomers exerted qualitatively similar effects at about 10 to 200 times higher concentrations. Correspondingly, the increase in potency of the racemic mixtures of the drugs was accompanied by increases in stereoselectivity. It is concluded that (-)-desmethoxyverapamil is the most potent stereoselective calcium antagonist of the verapamil type with respect to its effects on heart and vascular smooth muscle

Effects of (-)-desmethoxyverapamil on heart and vascular smooth muscle

Energy Technology Data Exchange (ETDEWEB)

Nawrath, H.; Raschack, M.

1987-09-01

(-)-Desmethoxyverapamil (also known as (-)-devapamil or (-)-D888) has been developed as a verapamil type radioligand for the study of calcium channels. In the present investigation, the effects of (-)-desmethoxyverapamil on action potential (AP) and force of contraction in heart muscle preparations and on tension and /sup 45/Ca influx in vascular smooth muscle are described. In part, the effects were compared with the (+)-isomer of desmethoxyverapamil and the isomers of both verapamil and methoxyverapamil. In atrial and/or ventricular heart muscle preparations from guinea pigs, cats and man, (-)-desmethoxyverapamil decreased the force of contraction and shortened the AP duration. Slow response APs were depressed, whereas dV/dtmax of phase 0 of the AP remained unchanged. The rank order of potency of the (-)-isomers was as follows: desmethoxyverapamil greater than methoxyverapamil greater than verapamil. Potassium-induced contractures and /sup 45/Ca influx were depressed by the (-)-isomers of desmethoxyverapamil, methoxyverapamil and verapamil in the same potency rank order as observed in heart muscle. The (+)-isomers exerted qualitatively similar effects at about 10 to 200 times higher concentrations. Correspondingly, the increase in potency of the racemic mixtures of the drugs was accompanied by increases in stereoselectivity. It is concluded that (-)-desmethoxyverapamil is the most potent stereoselective calcium antagonist of the verapamil type with respect to its effects on heart and vascular smooth muscle.

Deletion of Dicer in smooth muscle affects voiding pattern and reduces detrusor contractility and neuroeffector transmission.

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Mardjaneh Karbalaei Sadegh

Full Text Available MicroRNAs have emerged as important regulators of smooth muscle phenotype and may play important roles in pathogenesis of various smooth muscle related disease states. The aim of this study was to investigate the role of miRNAs for urinary bladder function. We used an inducible and smooth muscle specific Dicer knockout (KO mouse which resulted in significantly reduced levels of miRNAs, including miR-145, miR-143, miR-22, miR125b-5p and miR-27a, from detrusor preparations without mucosa. Deletion of Dicer resulted in a disturbed micturition pattern in vivo and reduced depolarization-induced pressure development in the isolated detrusor. Furthermore, electrical field stimulation revealed a decreased cholinergic but maintained purinergic component of neurogenic activation in Dicer KO bladder strips. The ultrastructure of detrusor smooth muscle cells was well maintained, and the density of nerve terminals was similar. Western blotting demonstrated reduced contents of calponin and desmin. Smooth muscle α-actin, SM22α and myocardin were unchanged. Activation of strips with exogenous agonists showed that depolarization-induced contraction was preferentially reduced; ATP- and calyculin A-induced contractions were unchanged. Quantitative real time PCR and western blotting demonstrated reduced expression of Cav1.2 (Cacna1c. It is concluded that smooth muscle miRNAs play an important role for detrusor contractility and voiding pattern of unrestrained mice. This is mediated in part via effects on expression of smooth muscle differentiation markers and L-type Ca(2+ channels in the detrusor.

Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

International Nuclear Information System (INIS)

Costa-Silva, Bruno; Coelho da Costa, Meline; Melo, Fernanda Rosene; Neves, Cynara Mendes; Alvarez-Silva, Marcio; Calloni, Giordano Wosgrau; Trentin, Andrea Goncalves

2009-01-01

The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effect was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells

Enhanced elastin synthesis and maturation in human vascular smooth muscle tissue derived from induced-pluripotent stem cells.

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Eoh, Joon H; Shen, Nian; Burke, Jacqueline A; Hinderer, Svenja; Xia, Zhiyong; Schenke-Layland, Katja; Gerecht, Sharon

2017-04-01

Obtaining vascular smooth muscle tissue with mature, functional elastic fibers is a key obstacle in tissue-engineered blood vessels. Poor elastin secretion and organization leads to a loss of specialization in contractile smooth muscle cells, resulting in over proliferation and graft failure. In this study, human induced-pluripotent stem cells (hiPSCs) were differentiated into early smooth muscle cells, seeded onto a hybrid poly(ethylene glycol) dimethacrylate/poly (l-lactide) (PEGdma-PLA) scaffold and cultured in a bioreactor while exposed to pulsatile flow, towards maturation into contractile smooth muscle tissue. We evaluated the effects of pulsatile flow on cellular organization as well as elastin expression and assembly in the engineered tissue compared to a static control through immunohistochemistry, gene expression and functionality assays. We show that culturing under pulsatile flow resulted in organized and functional hiPSC derived smooth muscle tissue. Immunohistochemistry analysis revealed hiPSC-smooth muscle tissue with robust, well-organized cells and elastic fibers and the supporting microfibril proteins necessary for elastic fiber assembly. Through qRT-PCR analysis, we found significantly increased expression of elastin, fibronectin, and collagen I, indicating the synthesis of necessary extracellular matrix components. Functionality assays revealed that hiPSC-smooth muscle tissue cultured in the bioreactor had an increased calcium signaling and contraction in response to a cholinergic agonist, significantly higher mature elastin content and improved mechanical properties in comparison to the static control. The findings presented here detail an effective approach to engineering elastic human vascular smooth muscle tissue with the functionality necessary for tissue engineering and regenerative medicine applications. Obtaining robust, mature elastic fibers is a key obstacle in tissue-engineered blood vessels. Human induced-pluripotent stem cells have

MURC deficiency in smooth muscle attenuates pulmonary hypertension.

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Nakanishi, Naohiko; Ogata, Takehiro; Naito, Daisuke; Miyagawa, Kotaro; Taniguchi, Takuya; Hamaoka, Tetsuro; Maruyama, Naoki; Kasahara, Takeru; Nishi, Masahiro; Matoba, Satoaki; Ueyama, Tomomi

2016-08-22

Emerging evidence suggests that caveolin-1 (Cav1) is associated with pulmonary arterial hypertension. MURC (also called Cavin-4) is a member of the cavin family, which regulates caveolar formation and functions together with caveolins. Here, we show that hypoxia increased Murc mRNA expression in the mouse lung, and that Murc-null mice exhibited attenuation of hypoxia-induced pulmonary hypertension (PH) accompanied by reduced ROCK activity in the lung. Conditional knockout mice lacking Murc in smooth muscle also resist hypoxia-induced PH. MURC regulates the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) through Rho/ROCK signalling. Cav1 suppresses RhoA activity in PASMCs, which is reversed by MURC. MURC binds to Cav1 and inhibits the association of Cav1 with the active form of Gα13, resulting in the facilitated association of the active form of Gα13 with p115RhoGEF. These results reveal that MURC has a function in the development of PH through modulating Rho/ROCK signalling.

β-adrenergic relaxation of smooth muscle: differences between cells and tissues

International Nuclear Information System (INIS)

Scheid, C.R.

1987-01-01

The present studies were carried out in an attempt to resolve the controversy about the Na + dependence of β-adrenergic relaxation in smooth muscle. Previous studies on isolated smooth muscle cells from the toad stomach had suggested that at least some of the actions of β-adrenergic agents, including a stimulatory effect on 45 Ca efflux, were dependent on the presence of a normal transmembrane Na + gradient. Studies by other investigators using tissues derived from mammalian sources had suggested that the relaxing effect of β-adrenergic agents was Na + independent. Uncertainty remained as to whether these discrepancies reflected differences between cells and tissues or differences between species. Thus, in the present studies, the authors utilized both tissues and cells from the same source, the stomach muscle of the toad Bufo marinus, and assessed the Na + dependence of β-adrenergic relaxation. They found that elimination of a normal Na + gradient abolished β-adrenergic relaxation of isolated cells. In tissues, however, similar manipulations had no effect on relaxation. The reasons for this discrepancy are unclear but do not appear to be attributable to changes in smooth muscle function following enzymatic dispersion. Thus the controversy concerning the mechanisms of β-adrenergic relaxation may reflect inherent differences between tissues and cells

The effects of inhaled corticosteroids on intrinsic responsiveness and histology of airways from infant monkeys exposed to house dust mite allergen and ozone

International Nuclear Information System (INIS)

Joad, Jesse P.; Kott, Kayleen S.; Bric, John M.; Schelegle, Edward S.; Gershwin, Laurel J.; Plopper, Charles G.; Peake, Janice L.; Pinkerton, Kent E.

2008-01-01

Inhaled corticosteroids (ICS) are recommended to treat infants with asthma, some with intermittent asthma. We previously showed that exposing infant monkeys to allergen/ozone resulted in asthma-like characteristics of their airways. We evaluated the effects of ICS on histology and intrinsic responsiveness of allergen/ozone-exposed and normal infant primate airways. Infant monkeys were exposed by inhalation to (1) filtered air and saline, (2) house dust mite allergen (HDMA) + ozone and saline, (3) filtered air and ICS (budesonide) or (4) HDMA + ozone and ICS. Allergen/ozone exposures started at 1 month and ICS at 3 months of age. At 6 months of age, methacholine-induced changes in luminal area of airways in proximal and distal lung slices were determined using videomicrometry, followed by histology of the same slices. Proximal airway responsiveness was increased by allergen/ozone and by ICS. Eosinophil profiles were increased by allergen/ozone in both proximal and distal airways, an effect that was decreased by ICS in distal airways. In both allergen/ozone- and air-exposed monkeys, ICS increased the number of alveolar attachments in distal airways, decreased mucin in proximal airways and decreased epithelial volume in both airways. ICS increased smooth muscle in air-exposed animals while decreasing it in allergen/ozone-exposed animals in both airways. In proximal airways, there was a small but significant positive correlation between smooth muscle and airway responsiveness, as well as between alveolar attachments and responsiveness. ICS change morphology and function in normal airways as well as allergen/ozone-exposed airways, suggesting that they should be reserved for infants with active symptoms

A new paradigm for the role of smooth muscle cells in the human cervix.

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Vink, Joy Y; Qin, Sisi; Brock, Clifton O; Zork, Noelia M; Feltovich, Helen M; Chen, Xiaowei; Urie, Paul; Myers, Kristin M; Hall, Timothy J; Wapner, Ronald; Kitajewski, Jan K; Shawber, Carrie J; Gallos, George

2016-10-01

Premature cervical remodeling resulting in spontaneous preterm birth may begin with premature failure or relaxation at the internal os (termed "funneling"). To date, we do not understand why the internal os fails or why funneling occurs in some cases of premature cervical remodeling. Although the human cervix is thought to be mostly collagen with minimal cellular content, cervical smooth muscle cells are present in the cervix and can cause cervical tissue contractility. To understand why the internal os relaxes or why funneling occurs in some cases of premature cervical remodeling, we sought to evaluate cervical smooth muscle cell content and distribution throughout human cervix and correlate if cervical smooth muscle organization influences regional cervical tissue contractility. Using institutional review board-approved protocols, nonpregnant women cervix, whole cervical slices were obtained from the internal os, midcervix, and external os and immunostained with smooth muscle actin. To correlate tissue structure with function, whole slices from the internal and external os were stimulated to contract with 1 μmol/L of oxytocin in organ baths. In separate samples, we tested if the cervix responds to a common tocolytic, nifedipine. Cervical slices from the internal os were treated with oxytocin alone or oxytocin + increasing doses of nifedipine to generate a dose response and half maximal inhibitory concentration. Student t test was used where appropriate. Cervical tissue was collected from 41 women. Immunohistochemistry showed cervical smooth muscle cells at the internal and external os expressed mature smooth muscle cell markers and contraction-associated proteins. The cervix exhibited a gradient of cervical smooth muscle cells. The area of the internal os contained 50-60% cervical smooth muscle cells that were circumferentially organized in the periphery of the stroma, which may resemble a sphincter-like pattern. The external os contained approximately 10

K+-induced alterations in airway muscle responsiveness to electrical field stimulation

International Nuclear Information System (INIS)

Murlas, C.; Ehring, G.; Suszkiw, J.; Sperelakis, N.

1986-01-01

We investigated possible pre- and postsynaptic effects of K+-induced depolarization on ferret tracheal smooth muscle (TSM) responsiveness to cholinergic stimulation. To assess electromechanical activity, cell membrane potential (Em) and tension (Tm) were simultaneously recorded in buffer containing 6, 12, 18, or 24 mM K+ before and after electrical field stimulation (EFS) or exogenous acetylcholine (ACh). In 6 mM K+, Em was -58.1 +/- 1.0 mV (mean +/- SE). In 12 mM K+, Em was depolarized to -52.3 +/- 0.9 mV, basal Tm did not change, and both excitatory junctional potentials and contractile responses to EFS at short stimulus duration were larger than in 6 mM K+. No such potentiation occurred at a higher K+, although resting Em and Tm increased progressively above 12 mM K+. The sensitivity of ferret TSM to exogenous ACh appeared unaffected by K+. To determine whether the hyperresponsiveness in 12 mM K+ was due, in part, to augmented ACh release from intramural airway nerves, experiments were done using TSM preparations incubated with [3H]choline to measure [3H]ACh release at rest and during EFS. Although resting [3H]ACh release increased progressively in higher K+, release evoked by EFS was maximal in 12 mM K+ and declined in higher concentrations. We conclude that small elevations in the extracellular K+ concentration augment responsiveness of the airways, by increasing the release of ACh both at rest and during EFS from intramural cholinergic nerve terminals. Larger increases in K+ appear to be inhibitory, possibly due to voltage-dependent effects that occur both pre- and postsynaptically

Calcium-sensitivity of smooth muscle contraction in the isolated ...

African Journals Online (AJOL)

sensitivity of smooth muscle contraction were studied in the isolated perfused rat tail artery, employing the activators noradrenaline (NA) (3ìM) sand potassium chloride (KC1) (100mM). Experiments were conduced in Ca2+ - buffered saline.

Synthetic smooth muscle in the outer blood plexus of the rhinarium skin of Lemur catta L.

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Elofsson, Rolf; Kröger, Ronald H H

2017-01-01

The skin of the lemur nose tip (rhinarium) has arterioles in the outer vascular plexus that are endowed with an unusual coat of smooth muscle cells. Comparison with the arterioles of the same area in a number of unrelated mammalians shows that the lemur pattern is unique. The vascular smooth muscle cells belong to the synthetic type. The function of synthetic smooth muscles around the terminal vessels in the lemur rhinarium is unclear but may have additional functions beyond regulation of vessel diameter.

Suppression of vascular smooth muscle cells' proliferation and ...

African Journals Online (AJOL)

This study aimed to determine the effects of valsartan on the proliferation and migration of isolated rat vascular smooth muscle cells (VSMCs) and the expression of phospho-p42/44 mitogen-activated protein kinase (MAPK) promoted by angiotensin II (Ang II). VSMCs from the rat thoracic aorta were cultured by ...

Prolonged ozone exposure in an allergic airway disease model: Adaptation of airway responsiveness and airway remodeling

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Park Chang-Soo

2006-02-01

Full Text Available Abstract Background Short-term exposure to high concentrations of ozone has been shown to increase airway hyper-responsiveness (AHR. Because the changes in AHR and airway inflammation and structure after chronic ozone exposure need to be determined, the goal of this study was to investigate these effects in a murine model of allergic airway disease. Methods We exposed BALB/c mice to 2 ppm ozone for 4, 8, and 12 weeks. We measured the enhanced pause (Penh to methacholine and performed cell differentials in bronchoalveolar lavage fluid. We quantified the levels of IL-4 and IFN-γ in the supernatants of the bronchoalveolar lavage fluids using enzyme immunoassays, and examined the airway architecture under light and electron microscopy. Results The groups exposed to ozone for 4, 8, and 12 weeks demonstrated decreased Penh at methacholine concentrations of 12.5, 25, and 50 mg/ml, with a dose-response curve to the right of that for the filtered-air group. Neutrophils and eosinophils increased in the group exposed to ozone for 4 weeks compared to those in the filtered-air group. The ratio of IL-4 to INF-γ increased significantly after exposure to ozone for 8 and 12 weeks compared to the ratio for the filtered-air group. The numbers of goblet cells, myofibroblasts, and smooth muscle cells showed time-dependent increases in lung tissue sections from the groups exposed to ozone for 4, 8, and 12 weeks. Conclusion These findings demonstrate that the increase in AHR associated with the allergic airway does not persist during chronic ozone exposure, indicating that airway remodeling and adaptation following repeated exposure to air pollutants can provide protection against AHR.

The effect of sleep onset on upper airway muscle activity in patients with sleep apnoea versus controls

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Fogel, Robert B; Trinder, John; White, David P; Malhotra, Atul; Raneri, Jill; Schory, Karen; Kleverlaan, Darci; Pierce, Robert J

2005-01-01

Pharyngeal dilator muscles are important in the pathophysiology of obstructive sleep apnoea syndrome (OSA). We have previously shown that during wakefulness, the activity of both the genioglossus (GGEMG) and tensor palatini (TPEMG) is greater in patients with OSA compared with controls. Further, EMG activity decreases at sleep onset, and the decrement is greater in apnoea patients than in healthy controls. In addition, it is known that the prevalence of OSA is greater in middle-aged compared with younger men. Thus, we had two goals in this study. First we compared upper airway muscle activity between young and middle-aged healthy men compared with men with OSA. We also explored the mechanisms responsible for the decrement in muscle activity at sleep onset in these groups. We investigated muscle activity, ventilation , and upper airway resistance (UAR) during wakefulness and sleep onset (transition from α to θ EEG activity) in all three groups. Measurements were obtained during basal breathing (BB) and nasal continuous positive airway pressure (CPAP) was applied to reduce negative pressure-mediated muscle activation). We found that during wakefulness there was a gradation of GGEMG and UAR (younger < older < OSA) and that muscle activity was reduced by the application of nasal CPAP (to a greater degree in the OSA patients). Although CPAP eliminated differences in UAR during wakefulness and sleep, GGEMG remained greater in the OSA patients. During sleep onset, a greater initial fall in GGEMG was seen in the OSA patients followed by subsequent muscle recruitment in the third to fifth breaths following the α to θ transition. On the CPAP night, and GGEMG still fell further in the OSA patients compared with control subjects. CPAP prevented the rise in UAR at sleep onset along with the associated recruitment in GGEMG. Differences in TPEMG among the groups were not significant. These data suggest that the middle-aged men had upper airway function midway between that of

Suppression of the increasing level of acetylcholine-stimulated intracellular Ca2+ in guinea pig airway smooth muscle cells by mabuterol.

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Song, Xirui; Zhao, Chao; Dai, Cailing; Ren, Yanxin; An, Nan; Wen, Huimin; Pan, L I; Cheng, Maosheng; Zhang, Yuyang

2015-11-01

The present study aimed to establish an effective method for the in vitro culture of guinea pig airway smooth muscle (ASM) cells, and also investigate the suppressive effect of mabuterol hydrochloride (Mab) on the increased level of intracellular Ca 2+ in ASM cells induced with acetylcholine (Ach). Two different methods, i.e. with or without collagenase to pretreat tracheal tissues, were applied to the manufacture of ASM cells. Cell viability was determined with the 3-(4,5-dimethylthinazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Immunocytochemistry and immunofluorescence were used for the identification of ASM cells. Different concentration levels (10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 mmol/l) of Mab were administered 5 min before Ach (10 -4 M) treatment, respectively. The Ca 2+ fluorescent probe, Fura-2/AM or Fluo-3/AM were applied to the inspection of Ca 2+ fluorescent intensity with Varioskan Flash, immunocytometry systems and an inverted system microscope, respectively. The results showed that the fresh method, in which isolated tracheal tissues were previously treated with collagenase for 20 min, was more advantageous for the preparation of guinea pig ASM cells compared to when the enzyme was not used. The time for the ASM cells to initially migrate out of the 'tissue blocks' and the culture having to be generated due to the thick cell density was significantly less. On identification with immunocytochemistry or immunofluorescent staining, >95% of the cells were ASM cells. Mab (10 -3 -10 -7 mmol/l) significantly suppressed the elevation of intracellular Ca 2+ induced by Ach in a concentration-dependent manner. The inhibitory rates of intracellular Ca 2+ by different concentrations of Mab, from low to high, were 14.93, 24.73, 40.06, 48.54 and 57.13%, respectively, when Varioskan Flash was used for determination. In conclusion, this novel method has a shorter harvesting period for ASM cells. Mab can suppress the increasing level of intracellular Ca 2

Microtissues Enhance Smooth Muscle Differentiation and Cell Viability of hADSCs for Three Dimensional Bioprinting

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Jin Yipeng

2017-07-01

Full Text Available Smooth muscle differentiated human adipose derived stem cells (hADSCs provide a crucial stem cell source for urinary tissue engineering, but the induction of hADSCs for smooth muscle differentiation still has several issues to overcome, including a relatively long induction time and equipment dependence, which limits access to abundant stem cells within a short period of time for further application. Three-dimensional (3D bioprinting holds great promise in regenerative medicine due to its controllable construction of a designed 3D structure. When evenly mixed with bioink, stem cells can be spatially distributed within a bioprinted 3D structure, thus avoiding drawbacks such as, stem cell detachment in a conventional cell-scaffold strategy. Notwithstanding the advantages mentioned above, cell viability is often compromised during 3D bioprinting, which is often due to pressure during the bioprinting process. The objective of our study was to improve the efficiency of hADSC smooth muscle differentiation and cell viability of a 3D bioprinted structure. Here, we employed the hanging-drop method to generate hADSC microtissues in a smooth muscle inductive medium containing human transforming growth factor β1 and bioprinted the induced microtissues onto a 3D structure. After 3 days of smooth muscle induction, the expression of α-smooth muscle actin and smoothelin was higher in microtissues than in their counterpart monolayer cultured hADSCs, as confirmed by immunofluorescence and western blotting analysis. The semi-quantitative assay showed that the expression of α-smooth muscle actin (α-SMA was 0.218 ± 0.077 in MTs and 0.082 ± 0.007 in Controls; smoothelin expression was 0.319 ± 0.02 in MTs and 0.178 ± 0.06 in Controls. Induced MTs maintained their phenotype after the bioprinting process. Live/dead and cell count kit 8 assays showed that cell viability and cell proliferation in the 3D structure printed with microtissues were higher at all time

Aortic smooth muscle cell proteoglycan synthesis in relation to atherosclerosis

International Nuclear Information System (INIS)

Edwards, I.J.

1989-01-01

Proteoglycans (PG) are implicated in atherogenesis by their effects on tissue permeability and cell proliferation and their interaction with plasma low density lipoproteins. Using the pigeon model in which an atherosclerosis-susceptible (WC) and -resistant (SR) breed can be compared, PG synthesis by cultured aortic smooth muscle cells was examined by the use of [ 35 S]-sodium sulfate and [ 3 H]-serine or [ 3 H]-glucosamine as labeling precursors. In both SR and WC cells, the majority of newly synthesized PG were secreted into the media. Chondroitin sulfate (CS) PG and dermatan sulfate (DS) PG were the major PG produced. Total PG production was consistently lower in WC compared to SR cultures due in part to reduce PG synthesis but also to degradation of newly synthesized PG. Since increased DS-PG accompanines atherosclerosis progression, experiments were designed to test the hypothesis that macrophages modulate smooth muscle cell metabolism to cause increase DS-PG production. Cultured WC aortic smooth muscle cells were exposed to the media of cholesteryl ester-loaded pigeon peritoneal macrophages or a macrophage cell line P388D1 and the production of PG examined. Increasing concentration of conditioned media from both types of macrophages caused increased incorporation of 35 S-sulfate into secreted PG, but no change in cell-associated PG. Lipopolysaccharide activation of P388D1 cells enhanced the effect

Hydroxyapatite and Calcified Elastin Induce Osteoblast-like Differentiation in Rat Aortic Smooth Muscle Cells

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Lei, Yang; Sinha, Aditi; Nosoudi, Nasim; Grover, Ankit; Vyavahare, Naren

2014-01-01

Vascular calcification can be categorized into two different types. Intimal calcification related to atherosclerosis and elastin-specific medial arterial calcification (MAC). Osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) has been shown in both types; however, how this relates to initiation of vascular calcification is unclear. We hypothesize that the initial deposition of hydroxyapatite-like mineral in MAC occurs on degraded elastin first and that causes osteogenic transformation of VSMCs. To test this, rat aortic smooth muscle cells (RASMCs) were cultured on hydroxyapatite crystals and calcified aortic elastin. Using RT-PCR and specific protein assays, we demonstrate that RASMCs lose their smooth muscle lineage markers like alpha smooth muscle actin (SMA) and myosin heavy chain (MHC) and undergo chondrogenic/osteogenic transformation. This is indicated by an increase in the expression of typical chondrogenic proteins such as aggrecan, collagen type II alpha 1(Col2a1) and bone proteins such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, when calcified conditions are removed, cells return to their original phenotype. Our data supports the hypothesis that elastin degradation and calcification precedes VSMCs' osteoblast-like differentiation. PMID:24447384

Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes.

Science.gov (United States)

Subasinghe, Supundi; Unabia, Sharon; Barrow, Colin J; Mok, Su San; Aguilar, Marie-Isabel; Small, David H

2003-02-01

Accumulation of beta amyloid (Abeta) in the brain is central to the pathogenesis of Alzheimer's disease. Abeta can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Abeta binding to membranes. Abeta peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Abeta peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Abeta peptides and their membrane binding. 'Ageing' the Abeta peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Abeta analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Abeta to purified plasma membrane preparations but also reduced Abeta toxicity. The results support the view that Abeta toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Abeta-membrane binding.

Establishment of artery smooth muscle cell proliferation model after subarachnoid hemorrhage in rats

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Yu-jie CHEN

2011-12-01

Full Text Available Objective The current paper aims to simulate the effects of hemolytic products on intracranial vascular smooth muscle cell after subarachnoid hemorrhage(SAH,and probe into the molecular mechanism and strategy for the prevention and cure of vascular proliferation after SAH.Methods Thirty Sprague-Dawley rats were randomly divided into three groups,including sham-operated,24 h after SAH,and 72 h after SAH groups.The artificial hemorrhage model around the common carotid artery was established for the latter two groups.The animals were put to death after 24 h and 72 h to take the common carotid artery,and to measure the expression level of PCNA,SM-α-actin protein,and mRNA in the smooth muscle cell.Results The PCNA mRNA expression was significantly up-regulated in the 24-h group(P < 0.01.The expression in the 72-h group was lower than that of the 24-h group(P < 0.01,whereas it was still remarkably higher than that of the sham group(P < 0.01.The SM-α-actin mRNA expression in the smooth muscle cell in the 24-h and 72-h groups decreased compared with that of the Sham group(P < 0.05,whereas the 72-h group was significantly lower than that of the 24-h group(P < 0.05.The protein expression of PCNA and SM-α-actin showed a similar trend.Conclusion The current experiment simulates better effects of the hemolytic products on vascular smooth muscle cell after SAH.It also shows that artificial hemorrhage around the common carotid artery could stimulate vascular smooth muscle cell to change from contractile phenotype into synthetic phenotype,and improve it to proliferate.

Augmented vascular smooth muscle cell stiffness and adhesion when hypertension is superimposed on aging.

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Sehgel, Nancy L; Sun, Zhe; Hong, Zhongkui; Hunter, William C; Hill, Michael A; Vatner, Dorothy E; Vatner, Stephen F; Meininger, Gerald A

2015-02-01

Hypertension and aging are both recognized to increase aortic stiffness, but their interactions are not completely understood. Most previous studies have attributed increased aortic stiffness to changes in extracellular matrix proteins that alter the mechanical properties of the vascular wall. Alternatively, we hypothesized that a significant component of increased vascular stiffness in hypertension is due to changes in the mechanical and adhesive properties of vascular smooth muscle cells, and that aging would augment the contribution from vascular smooth muscle cells when compared with the extracellular matrix. Accordingly, we studied aortic stiffness in young (16-week-old) and old (64-week-old) spontaneously hypertensive rats and Wistar-Kyoto wild-type controls. Systolic and pulse pressures were significantly increased in young spontaneously hypertensive rats when compared with young Wistar-Kyoto rats, and these continued to rise in old spontaneously hypertensive rats when compared with age-matched controls. Excised aortic ring segments exhibited significantly greater elastic moduli in both young and old spontaneously hypertensive rats versus Wistar-Kyoto rats. were isolated from the thoracic aorta, and stiffness and adhesion to fibronectin were measured by atomic force microscopy. Hypertension increased both vascular smooth muscle cell stiffness and vascular smooth muscle cell adhesion, and these increases were both augmented with aging. By contrast, hypertension did not affect histological measures of aortic collagen and elastin, which were predominantly changed by aging. These findings support the concept that stiffness and adhesive properties of vascular smooth muscle cells are novel mechanisms contributing to the increased aortic stiffness occurring with hypertension superimposed on aging. © 2014 American Heart Association, Inc.

Role of SM22 in the differential regulation of phasic vs. tonic smooth muscle

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Ali, Mehboob

2015-01-01

Preliminary proteomics studies between tonic vs. phasic smooth muscles identified three distinct protein spots identified to be those of transgelin (SM22). The latter was found to be distinctly downregulated in the internal anal sphincter (IAS) vs. rectal smooth muscle (RSM) SMC. The major focus of the present studies was to examine the differential molecular control mechanisms by SM22 in the functionality of truly tonic smooth muscle of the IAS vs. the adjoining phasic smooth muscle of the RSM. We monitored SMC lengths before and after incubation with pFLAG-SM22 (for SM22 overexpression), and SM22 small-interfering RNA. pFLAG-SM22 caused concentration-dependent and significantly greater relaxation in the IAS vs. the RSM SMCs. Conversely, temporary silencing of SM22 caused contraction in both types of the SMCs. Further studies revealed a significant reverse relationship between the levels of SM22 phosphorylation and the amount of SM22-actin binding in the IAS and RSM SMC. Data showed higher phospho-SM22 levels and decreased SM22-actin binding in the IAS, and reverse to be the case in the RSM SMCs. Experiments determining the mechanism for SM22 phosphorylation in these smooth muscles revealed that Y-27632 (Rho kinase inhibitor) but not Gö-6850 (protein kinase C inhibitor) caused concentration-dependent decreased phosphorylation of SM22. We speculate that SM22 plays an important role in the regulation of basal tone via Rho kinase-induced phosphorylation of SM22. PMID:25617350

Voltage-dependent inward currents in smooth muscle cells of skeletal muscle arterioles

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Shirokov, Roman E.

2018-01-01

Voltage-dependent inward currents responsible for the depolarizing phase of action potentials were characterized in smooth muscle cells of 4th order arterioles in mouse skeletal muscle. Currents through L-type Ca2+ channels were expected to be dominant; however, action potentials were not eliminated in nominally Ca2+-free bathing solution or by addition of L-type Ca2+ channel blocker nifedipine (10 μM). Instead, Na+ channel blocker tetrodotoxin (TTX, 1 μM) reduced the maximal velocity of the upstroke at low, but not at normal (2 mM), Ca2+ in the bath. The magnitude of TTX-sensitive currents recorded with 140 mM Na+ was about 20 pA/pF. TTX-sensitive currents decreased five-fold when Ca2+ increased from 2 to 10 mM. The currents reduced three-fold in the presence of 10 mM caffeine, but remained unaltered by 1 mM of isobutylmethylxanthine (IBMX). In addition to L-type Ca2+ currents (15 pA/pF in 20 mM Ca2+), we also found Ca2+ currents that are resistant to 10 μM nifedipine (5 pA/pF in 20 mM Ca2+). Based on their biophysical properties, these Ca2+ currents are likely to be through voltage-gated T-type Ca2+ channels. Our results suggest that Na+ and at least two types (T- and L-) of Ca2+ voltage-gated channels contribute to depolarization of smooth muscle cells in skeletal muscle arterioles. Voltage-gated Na+ channels appear to be under a tight control by Ca2+ signaling. PMID:29694371

Laboratory practical to study the differential innervation pathways of urinary tract smooth muscle.

Science.gov (United States)

Rembetski, Benjamin E; Cobine, Caroline A; Drumm, Bernard T

2018-06-01

In the mammalian lower urinary tract, there is a reciprocal relationship between the contractile state of the bladder and urethra. As the bladder fills with urine, it remains relaxed to accommodate increases in volume, while the urethra remains contracted to prevent leakage of urine from the bladder to the exterior. Disruptions to the normal contractile state of the bladder and urethra can lead to abnormal micturition patterns and urinary incontinence. While both the bladder and urethra are smooth-muscle organs, they are differentially contracted by input from cholinergic and sympathetic nerves, respectively. The laboratory practical described here provides an experiential approach to understanding the anatomy of the lower urinary tract. Several key factors in urinary tract physiology are outlined, e.g., the bladder is contracted by activation of the parasympathetic pathway via cholinergic stimulation on muscarinic receptors, whereas the urethra is contracted by activation of the sympathetic pathway via adrenergic stimulation on α 1 -adrenoceptors. This is achieved by measuring the force generated by bladder and urethra smooth muscle to demonstrate that acetylcholine contracts the smooth muscle of the bladder, whereas adrenergic agonists contract the urethral smooth muscle. An inhibition of these effects is also demonstrated by application of the muscarinic receptor antagonist atropine and the α 1 -adrenergic receptor blocker phentolamine. A list of suggested techniques and exam questions to evaluate student understanding on this topic is also provided.

ADAMTS9-Regulated Pericellular Matrix Dynamics Governs Focal Adhesion-Dependent Smooth Muscle Differentiation

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Timothy J. Mead

2018-04-01

Full Text Available Summary: Focal adhesions anchor cells to extracellular matrix (ECM and direct assembly of a pre-stressed actin cytoskeleton. They act as a cellular sensor and regulator, linking ECM to the nucleus. Here, we identify proteolytic turnover of the anti-adhesive proteoglycan versican as a requirement for maintenance of smooth muscle cell (SMC focal adhesions. Using conditional deletion in mice, we show that ADAMTS9, a secreted metalloprotease, is required for myometrial activation during late gestation and for parturition. Through knockdown of ADAMTS9 in uterine SMC, and manipulation of pericellular versican via knockdown or proteolysis, we demonstrate that regulated pericellular matrix dynamics is essential for focal adhesion maintenance. By influencing focal adhesion formation, pericellular versican acts upstream of cytoskeletal assembly and SMC differentiation. Thus, pericellular versican proteolysis by ADAMTS9 balances pro- and anti-adhesive forces to maintain an SMC phenotype, providing a concrete example of the dynamic reciprocity of cells and their ECM. : Mead et al. identify a proteolytic mechanism that actively maintains a pericellular microenvironment conducive to uterine smooth muscle activation prior to parturition. They show that pericellular matrix proteolysis by the secreted metalloprotease ADAMTS9 is crucial for maintenance of focal adhesions in uterine smooth muscle cells, and its absence impairs parturition. Keywords: metalloprotease, extracellular matrix, smooth muscle, proteoglycan, myometrium, parturition, uterus, focal adhesion, proteolysis, interference reflection microscopy

KCl cotransport regulation and protein kinase G in cultured vascular smooth muscle cells.

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Adragna, N C; Zhang, J; Di Fulvio, M; Lincoln, T M; Lauf, P K

2002-05-15

K-Cl cotransport is activated by vasodilators in erythrocytes and vascular smooth muscle cells and its regulation involves putative kinase/phosphatase cascades. N-ethylmaleimide (NEM) activates the system presumably by inhibiting a protein kinase. Nitrovasodilators relax smooth muscle via cGMP-dependent activation of protein kinase G (PKG), a regulator of membrane channels and transporters. We investigated whether PKG regulates K-Cl cotransport activity or mRNA expression in normal, PKG-deficient-vector-only-transfected (PKG-) and PKG-catalytic-domain-transfected (PKG+) rat aortic smooth muscle cells. K-Cl cotransport was calculated as the Cl-dependent Rb influx, and mRNA was determined by semiquantitative RT-PCR. Baseline K-Cl cotransport was higher in PKG+ than in PKG- cells (p <0.01). At 0.5 mM, NEM stimulated K-Cl cotransport by 5-fold in PKG- but not in PKG+ cells. However, NEM was more potent although less effective to activate K-Cl cotransport in normal (passage 1-3) and PKG+ than in PKG- cells. In PKG- cells, [(dihydroindenyl) oxy] alkanoic acid (300 mM) but not furosemide (1 mM) inhibited K-Cl cotransport. Furthermore, no difference in K-Cl cotransport mRNA expression was observed between these cells. In conclusion, this study shows that manipulation of PKG expression in vascular smooth muscle cells affects K-Cl cotransport activity and its activation by NEM.

Smooth Muscle Endothelin B Receptors Regulate Blood Pressure but Not Vascular Function or Neointimal Remodeling.

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Miller, Eileen; Czopek, Alicja; Duthie, Karolina M; Kirkby, Nicholas S; van de Putte, Elisabeth E Fransen; Christen, Sibylle; Kimmitt, Robert A; Moorhouse, Rebecca; Castellan, Raphael F P; Kotelevtsev, Yuri V; Kuc, Rhoda E; Davenport, Anthony P; Dhaun, Neeraj; Webb, David J; Hadoke, Patrick W F

2017-02-01

The role of smooth muscle endothelin B (ET B ) receptors in regulating vascular function, blood pressure (BP), and neointimal remodeling has not been established. Selective knockout mice were generated to address the hypothesis that loss of smooth muscle ET B receptors would reduce BP, alter vascular contractility, and inhibit neointimal remodeling. ET B receptors were selectively deleted from smooth muscle by crossing floxed ET B mice with those expressing cre-recombinase controlled by the transgelin promoter. Functional consequences of ET B deletion were assessed using myography. BP was measured by telemetry, and neointimal lesion formation induced by femoral artery injury. Lesion size and composition (day 28) were analyzed using optical projection tomography, histology, and immunohistochemistry. Selective deletion of ET B was confirmed by genotyping, autoradiography, polymerase chain reaction, and immunohistochemistry. ET B -mediated contraction was reduced in trachea, but abolished from mesenteric veins, of knockout mice. Induction of ET B -mediated contraction in mesenteric arteries was also abolished in these mice. Femoral artery function was unaltered, and baseline BP modestly elevated in smooth muscle ET B knockout compared with controls (+4.2±0.2 mm Hg; P<0.0001), but salt-induced and ET B blockade-mediated hypertension were unaltered. Circulating endothelin-1 was not altered in knockout mice. ET B -mediated contraction was not induced in femoral arteries by incubation in culture medium or lesion formation, and lesion size was not altered in smooth muscle ET B knockout mice. In the absence of other pathology, ET B receptors in vascular smooth muscle make a small but significant contribution to ET B -dependent regulation of BP. These ET B receptors have no effect on vascular contraction or neointimal remodeling. © 2016 The Authors.

Intermediate filaments in smooth muscle from pregnant and non-pregnant human uterus.

OpenAIRE

Leoni, P; Carli, F; Halliday, D

1990-01-01

The intermediate filament proteins desmin and vimentin from pregnant and non-pregnant uterine muscle and smooth-muscle cells in culture were analysed using SDS/PAGE. The desmin content in uterine muscle increases dramatically during pregnancy, whereas vimentin remains unchanged or changes very little. When muscle cells are kept in culture, a considerable increase in vimentin content is observed as compared with vimentin in freshly isolated non-pregnant uterine tissue. Our results strengthen t...

Alcohol and airways function in health and disease.

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Sisson, Joseph H

2007-08-01

The volatility of alcohol promotes the movement of alcohol from the bronchial circulation across the airway epithelium and into the conducting airways of the lung. The exposure of the airways through this route likely accounts for many of the biologic effects of alcohol on lung airway functions. The effect of alcohol on lung airway functions is dependent on the concentration, duration, and route of exposure. Brief exposure to mild concentrations of alcohol may enhance mucociliary clearance, stimulates bronchodilation, and probably attenuates the airway inflammation and injury observed in asthma and chronic obstructive pulmonary disease (COPD). Prolonged and heavy exposure to alcohol impairs mucociliary clearance, may complicate asthma management, and likely worsens outcomes including lung function and mortality in COPD patients. Nonalcohol congeners and alcohol metabolites act as triggers for airway disease exacerbations especially in atopic asthmatics and in Asian populations who have a reduced capacity to metabolize alcohol. Research focused on the mechanisms of alcohol-mediated changes in airway functions has identified specific mechanisms that mediate alcohol effects within the lung airways. These include prominent roles for the second messengers calcium and nitric oxide, regulatory kinases including PKG and PKA, alcohol- and acetaldehyde-metabolizing enzymes such as aldehyde dehydrogenase 2. The role alcohol may play in the pathobiology of airway mucus, bronchial blood flow, airway smooth muscle regulation, and the interaction with other airway exposure agents, such as cigarette smoke, represents opportunities for future investigation.

Interstitial cells of Cajal in human small intestine. Ultrastructural identification and organization between the main smooth muscle layers

DEFF Research Database (Denmark)

Rumessen, Jüri Johannes; Thuneberg, Lars

1991-01-01

Anatomy, interstitial cells of Cajal, small intestine, gut motility, pacemaker cells, smooth muscle......Anatomy, interstitial cells of Cajal, small intestine, gut motility, pacemaker cells, smooth muscle...

Immunohistochemical characterization of endometriosis-associated smooth muscle cells in human peritoneal endometriotic lesions.

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Barcena de Arellano, Maria L; Gericke, Jessica; Reichelt, Uta; Okuducu, Ali Fuat; Ebert, Andreas D; Chiantera, Vito; Schneider, Achim; Mechsner, Sylvia

2011-10-01

Smooth muscle cells (SMC) are common components of endometriotic lesions. SMC have been characterized previously in peritoneal, ovarian and deep infiltrating endometriotic lesions and adenomyosis. The aim of this retrospective study was to investigate the extent of differentiation in endometriosis-associated SMC (EMaSMC) in peritoneal endometriotic lesions. We obtained biopsies from peritoneal endometriotic lesions (n = 60) and peritoneal sites distant from the endometriotic lesion (n = 60), as well as healthy peritoneum from patients without endometriosis (control tissue, n = 10). These controls were hysterectomy specimens from patients without endometriosis or adenomyosis. Histopathological examination of peritoneal specimens using antibodies against oxytocin receptor (OTR), vasopressin receptor (VPR), smooth muscle myosin heavy chain (SM-MHC), estrogen receptor (ER) or progesterone receptor (PR) was performed. To identify SMC and their level of differentiation, antibodies for smooth muscle actin desmin and caldesmon were used. SMC were detected in all endometriotic lesions. SMC were more abundant in unaffected peritoneum of women with endometriosis (38%) compared with women without endometriosis (6%; P endometriosis.

Smooth muscle relaxant activity of Crocus sativus (saffron and its constituents: possible mechanisms

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Amin Mokhtari-Zaer

2015-08-01

Full Text Available Saffron, Crocus sativus L. (C. sativus is rich in carotenoids and used in traditional medicine for treatment of various conditions such as coughs, stomach disorders, amenorrhea, asthma and cardiovascular disorders. These therapeutic effects of the plant are suggested to be due to its relaxant effect on smooth muscles. The effect of C. sativus and its constituents on different smooth muscles and the underlying mechanisms have been studied. Several studies have shown the relaxant effects of C. sativus and its constituents including safranal, crocin, crocetin and kaempferol on blood vessels. In addition, it was reported that saffron stigma lowers systolic blood pressure. The present review highlights the relaxant effects of C. sativus and its constituents on various smooth muscles. The possible mechanisms of this relaxing effect including activation of ß2-adrenoceptors, inhibition of histamine H1 and muscarinic receptors and calcium channels and modulation of nitric oxide (NO are also reviewed.

MicroRNA-20b-5p inhibits platelet-derived growth factor-induced proliferation of human fetal airway smooth muscle cells by targeting signal transducer and activator of transcription 3.

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Tang, Jin; Luo, Lingying

2018-06-01

Pediatric asthma is still a health threat to the pediatric population in recent years. The airway remodeling induced by abnormal airway smooth muscle (ASM) cell proliferation is an important cause of asthma. MicroRNAs (miRNAs) are important regulators of ASM cell proliferation. Numerous studies have reported that miR-20b-5p is a critical regulator for cell proliferation. However, whether miR-20b-5p is involved in regulating ASM cell proliferation remains unknown. In this study, we aimed to investigate the potential role of miR-20b-5p in regulating the proliferation of fetal ASM cell induced by platelet-derived growth factor (PDGF). Here, we showed that miR-20b-5p was significantly decreased in fetal ASM cells treated with PDGF. Biological experiments showed that the overexpression of miR-20b-5p inhibited the proliferation while miR-20b-5p inhibition markedly promoted the proliferation of fetal ASM cells. Bioinformatics analysis and luciferase reporter assay showed that miR-20b-5p directly targeted the 3'-UTR of signal transducer and activator of transcription 3 (STAT3). Further data showed that miR-20b-5p negatively regulated the expression of STAT3 in fetal ASM cells. Moreover, miR-20b-5p regulates the transcriptional activity of STAT3 in fetal ASM cells. Overexpression of STAT3 reversed the inhibitory effect of miR-20b-5p overexpression on fetal ASM cell proliferation while the knockdown of STAT3 abrogated the promoted effect of miR-20b-5p inhibition on fetal ASM cell proliferation. Overall, our results show that miR-20b-5p impedes PDGF-induced proliferation of fetal ASM cells through targeting STAT3. Our study suggests that miR-20b-5p may play an important role in airway remodeling during asthma and suggests that miR-20b-5p may serve as a potential therapeutic target for pediatric asthma. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Impact of Persistent Intracellular Infections on the Processes of Airway Remodeling in Children with Bronchial Asthma

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O.Ye. Chernyshova

2015-03-01

Full Text Available The article presents information about the impact of persistent intracellular infections on the processes of airway remodeling in bronchial asthma in children. The influence of matrix metalloproteinases, tissue inhibitor of matrix metalloproteinase, transforming growth factor, antibodies to type III collagen, endothelin-1 on the processes of morphological reconstruction of the airway by way of smooth muscle hypertrophy, enhanced neovascularization, epithelial cell hyperplasia, collagen deposition, thickening of the basal membrane, observed in bronchial asthma in children, were described.

CHARACTERIZATION OF TIGHTLY-ASSOCIATED SMOOTH MUSCLE MYOSIN-MYOSIN LIGHT CHAIN KINASE-CALMODULIN COMPLEXES*

OpenAIRE

Hong, Feng; Haldeman, Brian D.; John, Olivia A.; Brewer, Paul D.; Wu, Yi-Ying; Ni, Shaowei; Wilson, David P.; Walsh, Michael P.; Baker, Jonathan E.; Cremo, Christine R.

2009-01-01

A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by smooth muscle myosin light chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM (up-SMM) from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73 ± 9), the ratio was ~ 23–37% of that in gizzard tissue. Fifteen t...

MicroRNAs dynamically remodel gastrointestinal smooth muscle cells.

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Chanjae Park

2011-04-01

Full Text Available Smooth muscle cells (SMCs express a unique set of microRNAs (miRNAs which regulate and maintain the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal (GI SMCs in a transgenic animal model. We generated SMC-specific Dicer null animals that express the reporter, green fluorescence protein, in a SMC-specific manner. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs: the mutant mice developed severe dilation of the intestinal tract associated with the thinning and destruction of the smooth muscle (SM layers; contractile motility in the mutant intestine was dramatically decreased; and SM contractile genes and transcriptional regulators were extensively down-regulated in the mutant SMCs. Profiling and bioinformatic analyses showed that SMC phenotype is regulated by a complex network of positive and negative feedback by SMC miRNAs, serum response factor (SRF, and other transcriptional factors. Taken together, our data suggest that SMC miRNAs are required for the development and survival of SMCs in the GI tract.

Biophysical induction of vascular smooth muscle cell podosomes.

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Na Young Kim

Full Text Available Vascular smooth muscle cell (VSMC migration and matrix degradation occurs with intimal hyperplasia associated with atherosclerosis, vascular injury, and restenosis. One proposed mechanism by which VSMCs degrade matrix is through the use of podosomes, transient actin-based structures that are thought to play a role in extracellular matrix degradation by creating localized sites of matrix metalloproteinase (MMP secretion. To date, podosomes in VSMCs have largely been studied by stimulating cells with phorbol esters, such as phorbol 12,13-dibutyrate (PDBu, however little is known about the physiological cues that drive podosome formation. We present the first evidence that physiological, physical stimuli mimicking cues present within the microenvironment of diseased arteries can induce podosome formation in VSMCs. Both microtopographical cues and imposed pressure mimicking stage II hypertension induce podosome formation in A7R5 rat aortic smooth muscle cells. Moreover, wounding using a scratch assay induces podosomes at the leading edge of VSMCs. Notably the effect of each of these biophysical stimuli on podosome stimulation can be inhibited using a Src inhibitor. Together, these data indicate that physical cues can induce podosome formation in VSMCs.

Arterial wall mechanics as a function of heart rate: role of vascular smooth muscle

International Nuclear Information System (INIS)

Salvucci, Fernando Pablo; Schiavone, Jonathan; Craiem, Damian; Barra, Juan Gabriel

2007-01-01

Vascular wall viscoelasticity can be evaluated using a first-order lumped model. This model consists of a spring with elastic constant E and a dashpot with viscous constant η. More importantly, this viscoelastic model can be fitted in-vivo measuring arterial pressure and diameter. The aim of this work is to analyze the influence of heart rate over E and η. In two anesthetized sheep, diameter in thoracic aorta and intravascular pressure has been registered. The right atrium was connected to a programmable stimulator through a pair of pace-maker wires to produce changes in stimulation heart rate (HR) from 80 to 160 bpm. Additionally, local activation of vascular smooth muscle was induced with phenylephrine. After converting pressure and diameter signals into stress and strain respectively, E y η were calculated in control state and during muscle activation. The elastic modulus E did not present significant changes with heart rate. The viscous modulus η decreased 49% with a two-fold acceleration in heart rate from 80 to 160 bpm. However, the product η HR remained stable. The viscous modulus η increased 39% with smooth muscle activation. No significant pressure changes were registered during the experiment. The contractile action of vascular smooth muscle could contribute to increasing arterial wall viscosity. The decrease of η when HR increased might be related to smooth muscle relaxation mediated by endothelium activity, which was stimulated by flow increase. We conclude that HR can modulate arterial wall viscoelasticity through endothelium-dependent mechanisms

Effects of One Resistance Exercise Session on Vascular Smooth Muscle of Hypertensive Rats

International Nuclear Information System (INIS)

Silva, Tharciano Luiz Teixeira Braga da; Mota, Marcelo Mendonça; Fontes, Milene Tavares; Araújo, João Eliakim dos Santos; Carvalho, Vitor Oliveira; Bonjardim, Leonardo Rigoldi; Santos, Márcio Roberto Viana

2015-01-01

Hypertension is a public health problem and increases the incidence of cardiovascular diseases. To evaluate the effects of a resistance exercise session on the contractile and relaxing mechanisms of vascular smooth muscle in mesenteric arteries of N G -nitro L-arginine methyl ester (L-NAME)-induced hypertensive rats. Wistar rats were divided into three groups: control (C), hypertensive (H), and exercised hypertensive (EH). Hypertension was induced by administration of 20 mg/kg of L-NAME for 7 days prior to experimental protocols. The resistance exercise protocol consisted of 10 sets of 10 repetitions and intensity of 40% of one repetition maximum. The reactivity of vascular smooth muscle was evaluated by concentration‑response curves to phenylephrine (PHEN), potassium chloride (KCl) and sodium nitroprusside (SNP). Rats treated with L-NAME showed an increase (p < 0.001) in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) compared to the initial period of induction. No difference in PHEN sensitivity was observed between groups H and EH. Acute resistance exercise reduced (p < 0.001) the contractile response induced by KCl at concentrations of 40 and 60 mM in group EH. Greater (p < 0.01) smooth muscle sensitivity to NPS was observed in group EH as compared to group H. One resistance exercise session reduces the contractile response induced by KCl in addition to increasing the sensitivity of smooth muscle to NO in mesenteric arteries of hypertensive rats

Effects of One Resistance Exercise Session on Vascular Smooth Muscle of Hypertensive Rats

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Silva, Tharciano Luiz Teixeira Braga da; Mota, Marcelo Mendonça; Fontes, Milene Tavares; Araújo, João Eliakim dos Santos; Carvalho, Vitor Oliveira; Bonjardim, Leonardo Rigoldi; Santos, Márcio Roberto Viana, E-mail: marciorvsantos@bol.com.br [Universidade Federal de Sergipe, Universidade de São Paulo (Brazil)

2015-08-15

Hypertension is a public health problem and increases the incidence of cardiovascular diseases. To evaluate the effects of a resistance exercise session on the contractile and relaxing mechanisms of vascular smooth muscle in mesenteric arteries of N{sup G}-nitro L-arginine methyl ester (L-NAME)-induced hypertensive rats. Wistar rats were divided into three groups: control (C), hypertensive (H), and exercised hypertensive (EH). Hypertension was induced by administration of 20 mg/kg of L-NAME for 7 days prior to experimental protocols. The resistance exercise protocol consisted of 10 sets of 10 repetitions and intensity of 40% of one repetition maximum. The reactivity of vascular smooth muscle was evaluated by concentration‑response curves to phenylephrine (PHEN), potassium chloride (KCl) and sodium nitroprusside (SNP). Rats treated with L-NAME showed an increase (p < 0.001) in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) compared to the initial period of induction. No difference in PHEN sensitivity was observed between groups H and EH. Acute resistance exercise reduced (p < 0.001) the contractile response induced by KCl at concentrations of 40 and 60 mM in group EH. Greater (p < 0.01) smooth muscle sensitivity to NPS was observed in group EH as compared to group H. One resistance exercise session reduces the contractile response induced by KCl in addition to increasing the sensitivity of smooth muscle to NO in mesenteric arteries of hypertensive rats.

Effects of One Resistance Exercise Session on Vascular Smooth Muscle of Hypertensive Rats

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Tharciano Luiz Teixeira Braga da Silva

2015-01-01

Full Text Available Abstract Background: Hypertension is a public health problem and increases the incidence of cardiovascular diseases. Objective: To evaluate the effects of a resistance exercise session on the contractile and relaxing mechanisms of vascular smooth muscle in mesenteric arteries of NG-nitro L-arginine methyl ester (L-NAME-induced hypertensive rats. Methods: Wistar rats were divided into three groups: control (C, hypertensive (H, and exercised hypertensive (EH. Hypertension was induced by administration of 20 mg/kg of L-NAME for 7 days prior to experimental protocols. The resistance exercise protocol consisted of 10 sets of 10 repetitions and intensity of 40% of one repetition maximum. The reactivity of vascular smooth muscle was evaluated by concentration‑response curves to phenylephrine (PHEN, potassium chloride (KCl and sodium nitroprusside (SNP. Results: Rats treated with L-NAME showed an increase (p < 0.001 in systolic blood pressure (SBP, diastolic blood pressure (DBP and mean arterial pressure (MAP compared to the initial period of induction. No difference in PHEN sensitivity was observed between groups H and EH. Acute resistance exercise reduced (p < 0.001 the contractile response induced by KCl at concentrations of 40 and 60 mM in group EH. Greater (p < 0.01 smooth muscle sensitivity to NPS was observed in group EH as compared to group H. Conclusion: One resistance exercise session reduces the contractile response induced by KCl in addition to increasing the sensitivity of smooth muscle to NO in mesenteric arteries of hypertensive rats.

The potassium channel KCa3.1 as new therapeutic target for the prevention of obliterative airway disease

DEFF Research Database (Denmark)

Hua, Xiaoqin; Deuse, Tobias; Chen, Yi-Je

2013-01-01

The calcium-activated potassium channel KCa3.1 is critically involved in T-cell activation as well as in the proliferation of smooth muscle cells and fibroblasts. We sought to investigate whether KCa3.1 contributes to the pathogenesis of obliterative airway disease (OAD) and whether knockout or p...

Characterization of the effect of penehyclidine hydrochloride on muscarinic receptor subtypes mediating the contraction of guinea-pig isolated gastrointestinal smooth muscle.

Science.gov (United States)

Xiao, Hong-Tao; Liao, Zhi; Meng, Xian-Min; Yan, Xiao-Yan; Chen, Shu-Jie; Mo, Zheng-Ji

2009-07-01

The aim was to characterize the effect of penehyclidine hydrochloride, which mediates the relaxation of guinea-pig isolated gastrointestinal smooth muscle, on muscarinic receptor subtypes. Radioimmune assay was used to determine cAMP levels in isolated guinea-pig gastrointestinal smooth muscle to compare the selective effects of penehyclidine hydrochloride on muscarinic receptor subtypes. The results indicated that the relaxing effect of penehyclidine hydrochloride on isolated gastrointestinal smooth muscle contraction induced by acetylcholine was stronger than that of atropine (based on PA2 values). In the radioimmune assay, penehyclidine hydrochloride increased the cAMP content in isolated guinea-pig stomach smooth muscle and decreased the cAMP content in isolated guinea-pig intestinal smooth muscle, but the difference was not statistically significant at a dose of 10 mumol/l. The results suggest that penehyclidine hydrochloride has little or no effect on M2 receptor subtypes in guinea-pig gastrointestinal smooth muscle.

ASIC PROTEINS REGULATE SMOOTH MUSCLE CELL MIGRATION

OpenAIRE

Grifoni, Samira C.; Jernigan, Nikki L.; Hamilton, Gina; Drummond, Heather A.

2007-01-01

The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated Epithelial Na+ Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration, however the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence indi...

Bioenergetic profile of human coronary artery smooth muscle cells and effect of metabolic intervention.

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Mingming Yang

Full Text Available Bioenergetics of artery smooth muscle cells is critical in cardiovascular health and disease. An acute rise in metabolic demand causes vasodilation in systemic circulation while a chronic shift in bioenergetic profile may lead to vascular diseases. A decrease in intracellular ATP level may trigger physiological responses while dedifferentiation of contractile smooth muscle cells to a proliferative and migratory phenotype is often observed during pathological processes. Although it is now possible to dissect multiple building blocks of bioenergetic components quantitatively, detailed cellular bioenergetics of artery smooth muscle cells is still largely unknown. Thus, we profiled cellular bioenergetics of human coronary artery smooth muscle cells and effects of metabolic intervention. Mitochondria and glycolysis stress tests utilizing Seahorse technology revealed that mitochondrial oxidative phosphorylation accounted for 54.5% of ATP production at rest with the remaining 45.5% due to glycolysis. Stress tests also showed that oxidative phosphorylation and glycolysis can increase to a maximum of 3.5 fold and 1.25 fold, respectively, indicating that the former has a high reserve capacity. Analysis of bioenergetic profile indicated that aging cells have lower resting oxidative phosphorylation and reduced reserve capacity. Intracellular ATP level of a single cell was estimated to be over 1.1 mM. Application of metabolic modulators caused significant changes in mitochondria membrane potential, intracellular ATP level and ATP:ADP ratio. The detailed breakdown of cellular bioenergetics showed that proliferating human coronary artery smooth muscle cells rely more or less equally on oxidative phosphorylation and glycolysis at rest. These cells have high respiratory reserve capacity and low glycolysis reserve capacity. Metabolic intervention influences both intracellular ATP concentration and ATP:ADP ratio, where subtler changes may be detected by the latter.

Effect of an Ethanol Extract of Scutellaria baicalensis on Relaxation in Corpus Cavernosum Smooth Muscle

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Xiang Li

2012-01-01

Full Text Available Aims of study. The aim of the present study was to investigate whether an ethanol extract of Scutellaria baicalensis (ESB relaxes penile corpus cavernosum muscle in organ bath experiments. Materials and methods. Changes in tension of cavernous smooth muscle strips were determined by penile strip chamber model and in penile perfusion model. Isolated endothelium-intact rabbit corpus cavernosum was precontracted with phenylephrine (PE and then treated with ESB. Results. ESB relaxed penile smooth muscle in a dose-dependent manner, and this was inhibited by pre-treatment with NG-nitro-l-arginine methyl ester (l-NAME, a nitric oxide (NO synthase inhibitor, and 1H-[1, 2, 4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ, a soluble guanylyl cyclase (sGC inhibitor. ESB-induced relaxation was significantly attenuated by pretreatment with tetraethylammonium (TEA, a nonselective K+ channel blocker, and charybdotoxin, a selective Ca2+-dependent K+ channel inhibitor. ESB increased the cGMP levels of rabbit corpus cavernosum in a concentration-dependent manner without changes in cAMP levels. In a perfusion model of penile tissue, ESB also relaxed penile corpus cavernosum smooth muscle in a dose-dependent manner. Conclusion. Taken together, these results suggest that ESB relaxed rabbit cavernous smooth muscle via the NO/cGMP system and Ca2+-sensitive K+ channels in the corpus cavernosum.

Differentiation of Human Adipose Derived Stem Cells into Smooth Muscle Cells Is Modulated by CaMKIIγ

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Kaisaier Aji

2016-01-01

Full Text Available The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII is known to participate in maintenance and switches of smooth muscle cell (SMC phenotypes. However, which isoform of CaMKII is involved in differentiation of adult mesenchymal stem cells into contractile SMCs remains unclear. In the present study, we detected γ isoform of CaMKII in differentiation of human adipose derived stem cells (hASCs into SMCs that resulted from treatment with TGF-β1 and BMP4 in combination for 7 days. The results showed that CaMKIIγ increased gradually during differentiation of hASCs as determined by real-time PCR and western blot analysis. The siRNA-mediated knockdown of CaMKIIγ decreased the protein levels and transcriptional levels of smooth muscle contractile markers (a-SMA, SM22a, calponin, and SM-MHC, while CaMKIIγ overexpression increases the transcriptional and protein levels of smooth muscle contractile markers. These results suggested that γ isoform of CaMKII plays a significant role in smooth muscle differentiation of hASCs.

Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation

NARCIS (Netherlands)

Maarsingh, H; Tio, MA; Zaagsma, J; Meurs, H

2005-01-01

Background: Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO) production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using

Epigenetic regulation of vascular smooth muscle cell function in atherosclerosis.

Science.gov (United States)

Findeisen, Hannes M; Kahles, Florian K; Bruemmer, Dennis

2013-04-01

Epigenetics involve heritable and acquired changes in gene transcription that occur independently of the DNA sequence. Epigenetic mechanisms constitute a hierarchic upper-level of transcriptional control through complex modifications of chromosomal components and nuclear structures. These modifications include, for example, DNA methylation or post-translational modifications of core histones; they are mediated by various chromatin-modifying enzymes; and ultimately they define the accessibility of a transcriptional complex to its target DNA. Integrating epigenetic mechanisms into the pathophysiologic concept of complex and multifactorial diseases such as atherosclerosis may significantly enhance our understanding of related mechanisms and provide promising therapeutic approaches. Although still in its infancy, intriguing scientific progress has begun to elucidate the role of epigenetic mechanisms in vascular biology, particularly in the control of smooth muscle cell phenotypes. In this review, we will summarize epigenetic pathways in smooth muscle cells, focusing on mechanisms involved in the regulation of vascular remodeling.

Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells.

OpenAIRE

Marsh, K. A.; Hill, S. J.

1992-01-01

1. Bovine tracheal smooth muscle cells were established in culture to study agonist-induced phosphoinositide (PI) hydrolysis in this tissue. 2. Bradykinin (0.1 nM-10 microM) evoked a concentration-dependent increase (log EC50 (M) = -9.4 +/- 0.2; n = 8) in the accumulation of total [3H]-inositol phosphates in cultured tracheal smooth muscle cells whereas the selective B1 receptor agonist des-Arg9-bradykinin (10 microM) was significantly less effective (16% of bradykinin maximal response; relat...

Serelaxin Elicits Bronchodilation and Enhances β-Adrenoceptor-mediated Airway Relaxation

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Maggie Lam

2016-10-01

Full Text Available Treatment with β-adrenoceptor agonists does not fully overcome the symptoms associated with severe asthma. Serelaxin elicits potent uterine and vascular relaxation via its cognate receptor, RXFP1, and nitric oxide (NO signaling, and is being clinically evaluated for the treatment of acute heart failure. However, its direct bronchodilator efficacy has yet to be explored. Tracheal rings were prepared from male Sprague-Dawley rats (250-350g and tricolor guinea pigs, and precision cut lung slices (PCLS containing intrapulmonary airways were prepared from rats only. Recombinant human serelaxin (rhRLX alone and in combination with rosiglitazone (PPARγ agonist; recently described as a novel dilator or β-adrenoceptor agonists (isoprenaline, salbutamol were added either to pre-contracted airways, or before contraction with methacholine or endothelin-1. Regulation of rhRLX responses by epithelial removal, indomethacin (cyclooxygenase inhibitor, L-NAME (nitric oxide synthase inhibitor, SQ22536 (adenylate cyclase inhibitor and ODQ (guanylate cyclase inhibitor were also evaluated. Immunohistochemistry was used to localize RXFP1 to airway epithelium and smooth muscle. rhRLX elicited relaxation in rat trachea and PCLS, more slowly than rosiglitazone or isoprenaline, but potentiated relaxation to both these dilators. It markedly increased β-adrenoceptor agonist potency in guinea pig trachea. rhRLX, rosiglitazone and isoprenaline pretreatment also inhibited the development of rat tracheal contraction. Bronchoprotection by rhRLX increased with longer pre-incubation time, and was partially reduced by epithelial removal, indomethacin and/or L-NAME. SQ22536 and ODQ also partially inhibited rhRLX-mediated relaxation in both intact and epithelial-denuded trachea. RXFP1 expression in airway was at higher levels in epithelium than smooth muscle.In summary, rhRLX elicits large and small airway relaxation via epithelial-dependent and -independent mechanisms, likely

α-smooth muscle actin is not a marker of fibrogenic cell activity in skeletal muscle fibrosis.

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Wanming Zhao

Full Text Available α-Smooth muscle actin (α-SMA is used as a marker for a subset of activated fibrogenic cells, myofibroblasts, which are regarded as important effector cells of tissue fibrogenesis. We address whether α-SMA-expressing myofibroblasts are detectable in fibrotic muscles of mdx5cv mice, a mouse model for Duchenne muscular dystrophy (DMD, and whether the α-SMA expression correlates with the fibrogenic function of intramuscular fibrogenic cells. α-SMA immunostaining signal was not detected in collagen I (GFP-expressing cells in fibrotic muscles of ColI-GFP/mdx5cv mice, but it was readily detected in smooth muscle cells lining intramuscular blood vessel walls. α-SMA expression was detected by quantitative RT-PCR and Western blot in fibrogenic cells sorted from diaphragm and quadriceps muscles of the ColI-GFP/mdx5cv mice. Consistent with the more severe fibrosis in the ColI-GFP/mdx5cv diaphragm, the fibrogenic cells in the diaphragm exerted a stronger fibrogenic function than the fibrogenic cells in the quadriceps as gauged by their extracellular matrix gene expression. However, both gene and protein expression of α-SMA was lower in the diaphragm fibrogenic cells than in the quadriceps fibrogenic cells in the ColI-GFP/mdx5cv mice. We conclude that myofibroblasts are present in fibrotic skeletal muscles, but their expression of α-SMA is not detectable by immunostaining. The level of α-SMA expression by intramuscular fibrogenic cells does not correlate positively with the level of collagen gene expression or the severity of skeletal muscle fibrosis in the mdx5cv mice. α-SMA is not a functional marker of fibrogenic cells in skeletal muscle fibrosis associated with muscular dystrophy.

Smooth muscle cells of penis in the rat: noninvasive quantification with shear wave elastography.

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Zhang, Jia-Jie; Qiao, Xiao-Hui; Gao, Feng; Bai, Ming; Li, Fan; Du, Lian-Fang; Xing, Jin-Fang

2015-01-01

Smooth muscle cells (SMCs) of cavernosum play an important role in erection. It is of great significance to quantitatively analyze the level of SMCs in penis. In this study, we investigated the feasibility of shear wave elastography (SWE) on evaluating the level of SMCs in penis quantitatively. Twenty healthy male rats were selected. The SWE imaging of penis was carried out and then immunohistochemistry analysis of penis was performed to analyze the expression of alpha smooth muscle actin in penis. The measurement index of SWE examination was tissue stiffness (TS). The measurement index of immunohistochemistry analysis was positive area percentage of alpha smooth muscle actin (AP). Sixty sets of data of TS and AP were obtained. The results showed that TS was significantly correlated with AP and the correlation coefficient was -0.618 (p penis was successfully quantified in vivo with SWE. SWE can be used clinically for evaluating the level of SMCs in penis quantitatively.

Primary intraosseous smooth muscle tumor of uncertain malignant potential: original report and molecular characterization

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Lauren Kropp

2016-11-01

Full Text Available We report the first case of primary intraosseous smooth muscle tumor of uncertain malignant potential (STUMP which is analogous to borderline malignant uterine smooth muscle tumors so designated. The tumor presented in the femur of an otherwise healthy 30-year-old woman. Over a 3-year period, the patient underwent 11 biopsies or resections and 2 cytologic procedures. Multiple pathologists reviewed the histologic material including musculoskeletal pathologists but could not reach a definitive diagnosis. However, metastases eventually developed and were rapidly progressive and responsive to gemcitabine and docetaxel. Molecular characterization and ultrastructural analysis was consistent with smooth muscle origin, and amplification of unmutated chromosome 12p and 12q segments appears to be the major genomic driver of this tumor. Primary intraosseous STUMP is thought to be genetically related to leiomyosarcoma of bone, but likely representing an earlier stage of carcinogenesis. Wide excision and aggressive followup is warranted for this potentially life-threatening neoplasm.

Primary Intraosseous Smooth Muscle Tumor of Uncertain Malignant Potential: Original Report and Molecular Characterization.

Science.gov (United States)

Kropp, Lauren; Siegal, Gene P; Frampton, Garrett M; Rodriguez, Michael G; McKee, Svetlana; Conry, Robert M

2016-11-17

We report the first case of primary intraosseous smooth muscle tumor of uncertain malignant potential (STUMP) which is analogous to borderline malignant uterine smooth muscle tumors so designated. The tumor presented in the femur of an otherwise healthy 30-year-old woman. Over a 3-year period, the patient underwent 11 biopsies or resections and 2 cytologic procedures. Multiple pathologists reviewed the histologic material including musculoskeletal pathologists but could not reach a definitive diagnosis. However, metastases eventually developed and were rapidly progressive and responsive to gemcitabine and docetaxel. Molecular characterization and ultrastructural analysis was consistent with smooth muscle origin, and amplification of unmutated chromosome 12p and 12q segments appears to be the major genomic driver of this tumor. Primary intraosseous STUMP is thought to be genetically related to leiomyosarcoma of bone, but likely representing an earlier stage of carcinogenesis. Wide excision and aggressive follow-up is warranted for this potentially life-threatening neoplasm.

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

OpenAIRE

von Rechenberg Moritz; Peltier John M; Ahn Kwangmi; Zarembinski Thomas I; Askovich Peter S; An Steven S; Sahasrabudhe Sudhir; Fredberg Jeffrey J

2011-01-01

Abstract Background A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma. Methods Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two qua...

Effect of gamma rays on electrically evoked contractions of non-vascular smooth muscles (rat vas deferens)

International Nuclear Information System (INIS)

Azroony, R.; Ksies, F.; Alya, G.

2002-10-01

We have tried, in this experiment, to study the modifications of non-vascular smooth muscles contraction induced via gamma rays. Smooth muscular fibers were isolated from the vas deferens of an adult rat and contractions were electrically evoked. Our results show that irradiation activates the VOC (Voltage Operated Channel) type of ionic channels which causes an increasing in the inward flux of Ca 2+ and then causes an increasing in the inner calcium concentration [Ca 2] i, the matter which means an increasing in the force of muscular contraction. Concerning to the response of vas deferens smooth muscles to the activation of membrane receptors, we have tried to study the effects of gamma rays on activating adrenergic and cholinergic receptors, also, we have tried to show the effects of different doses of gamma rays (1, 3, 5, 7 Gy) on regulating the contractile response of this type of smooth muscles. And results show that: - Irradiation increases contraction force, mediated by adrenergic and cholinergic receptors, in a dose dependent manner, with E m ax 1 Gy m axc 3 Gy m ax 5 Gy m ax 7 Gy. There is an important shift on irradiated rats (3, 5, 7 Gy) where the maximum effect of Acetylcholine (E m ax) can be obtained in lower concentrations of Acetylcholine. These results mean that irradiation activates the inward flux of Ca 2+ through the ROC (Receptors Operated Channels) type of ionic channels, which rely, in their activation, on activating the membrane receptors. By comparing these results with the effects of gamma rays on activating vascular adrenergic and cholinergic receptors, we concluded that: Non-vascular smooth muscles (vas deferens) are less sensitive to irradiation in comparing with vascular smooth muscles (venae portal hepatica), and irradiation increases the sensitivity of cholinergic receptors to acetylcholine in the smooth muscular fibers of vas deferens while; if decreases this sensitivity in the smooth muscular fibers of venae portal hepatica

LRP1 in brain vascular smooth muscle cells mediates local clearance of Alzheimer's amyloid-β.

Science.gov (United States)

Kanekiyo, Takahisa; Liu, Chia-Chen; Shinohara, Mitsuru; Li, Jie; Bu, Guojun

2012-11-14

Impaired clearance of amyloid-β (Aβ) is a major pathogenic event for Alzheimer's disease (AD). Aβ depositions in brain parenchyma as senile plaques and along cerebrovasculature as cerebral amyloid angiopathy (CAA) are hallmarks of AD. A major pathway that mediates brain Aβ clearance is the cerebrovascular system where Aβ is eliminated through the blood-brain barrier (BBB) and/or degraded by cerebrovascular cells along the interstitial fluid drainage pathway. An Aβ clearance receptor, the low-density lipoprotein receptor-related protein 1 (LRP1), is abundantly expressed in cerebrovasculature, in particular in vascular smooth muscle cells. Previous studies have indicated a role of LRP1 in endothelial cells in transcytosing Aβ out of the brain across the BBB; however, whether this represents a significant pathway for brain Aβ clearance remains controversial. Here, we demonstrate that Aβ can be cleared locally in the cerebrovasculature by an LRP1-dependent endocytic pathway in smooth muscle cells. The uptake and degradation of both endogenous and exogenous Aβ were significantly reduced in LRP1-suppressed human brain vascular smooth muscle cells. Conditional deletion of Lrp1 in vascular smooth muscle cell in amyloid model APP/PS1 mice accelerated brain Aβ accumulation and exacerbated Aβ deposition as amyloid plaques and CAA without affecting Aβ production. Our results demonstrate that LRP1 is a major Aβ clearance receptor in cerebral vascular smooth muscle cell and a disturbance of this pathway contributes to Aβ accumulation. These studies establish critical functions of the cerebrovasculature system in Aβ metabolism and identify a new pathway involved in the pathogenesis of both AD and CAA.

Numerical Simulation for Mechanism of Airway Narrowing in Asthma

Science.gov (United States)

Bando, Kiyoshi; Yamashita, Daisuke; Ohba, Kenkichi

A calculation model is proposed to examine the generation mechanism of the numerous lobes on the inner-wall of the airway in asthmatic patients and to clarify luminal occlusion of the airway inducing breathing difficulties. The basement membrane in the airway wall is modeled as a two-dimensional thin-walled shell having inertia force due to the mass, and the smooth muscle contraction effect is replaced by uniform transmural pressure applied to the basement membrane. A dynamic explicit finite element method is used as a numerical simulation method. To examine the validity of the present model, simulation of an asthma attack is performed. The number of lobes generated in the basement membrane increases when transmural pressure is applied in a shorter time period. When the remodeling of the basement membrane occurs characterized by thickening and hardening, it is demonstrated that the number of lobes decreases and the narrowing of the airway lumen becomes severe. Comparison of the results calculated by the present model with those measured for animal experiments of asthma will be possible.

Kir2.1 regulates rat smooth muscle cell proliferation, migration, and post-injury carotid neointimal formation

International Nuclear Information System (INIS)

Qiao, Yong; Tang, Chengchun; Wang, Qingjie; Wang, Dong; Yan, Gaoliang; Zhu, Boqian

2016-01-01

Phenotype switching of vascular smooth muscle cells (VSMC) from the contractile type to the synthetic type is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. Inward rectifier K"+ channel 2.1 (Kir2.1) has been identified in VSMC. However, whether it plays a functional role in regulating cellular transformation remains obscure. In this study, we evaluated the role of Kir2.1 on VSMC proliferation, migration, phenotype switching, and post-injury carotid neointimal formation. Kir2.1 knockdown significantly suppressed platelet-derived growth factor BB-stimulated rat vascular smooth muscle cells (rat-VSMC) proliferation and migration. Deficiency in Kir2.1 contributed to the restoration of smooth muscle α-actin, smooth muscle 22α, and calponin and to a reduction in osteopontin expression in rat-VSMC. Moreover, the in vivo study showed that rat-VSMC switched to proliferative phenotypes and that knockdown of Kir2.1 significantly inhibited neointimal formation after rat carotid injury. Kir2.1 may be a potential therapeutic target in the treatment of cardiovascular diseases, such as atherosclerosis and restenosis following percutaneous coronary intervention.

Kir2.1 regulates rat smooth muscle cell proliferation, migration, and post-injury carotid neointimal formation

Energy Technology Data Exchange (ETDEWEB)

Qiao, Yong; Tang, Chengchun, E-mail: tangchengchun@medmail.com.cn; Wang, Qingjie; Wang, Dong; Yan, Gaoliang; Zhu, Boqian

2016-09-02

Phenotype switching of vascular smooth muscle cells (VSMC) from the contractile type to the synthetic type is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. Inward rectifier K{sup +} channel 2.1 (Kir2.1) has been identified in VSMC. However, whether it plays a functional role in regulating cellular transformation remains obscure. In this study, we evaluated the role of Kir2.1 on VSMC proliferation, migration, phenotype switching, and post-injury carotid neointimal formation. Kir2.1 knockdown significantly suppressed platelet-derived growth factor BB-stimulated rat vascular smooth muscle cells (rat-VSMC) proliferation and migration. Deficiency in Kir2.1 contributed to the restoration of smooth muscle α-actin, smooth muscle 22α, and calponin and to a reduction in osteopontin expression in rat-VSMC. Moreover, the in vivo study showed that rat-VSMC switched to proliferative phenotypes and that knockdown of Kir2.1 significantly inhibited neointimal formation after rat carotid injury. Kir2.1 may be a potential therapeutic target in the treatment of cardiovascular diseases, such as atherosclerosis and restenosis following percutaneous coronary intervention.

Inhibition of airway epithelial-to-mesenchymal transition and fibrosis by kaempferol in endotoxin-induced epithelial cells and ovalbumin-sensitized mice.

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Gong, Ju-Hyun; Cho, In-Hee; Shin, Daekeun; Han, Seon-Young; Park, Sin-Hye; Kang, Young-Hee

2014-03-01

Chronic airway remodeling is characterized by structural changes within the airway wall, including smooth muscle hypertrophy, submucosal fibrosis and epithelial shedding. Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis, which can be induced by TGF-β. In the in vitro study, we investigated whether 1-20 μM kaempferol inhibited lipopolysaccharide (LPS)-induced bronchial EMT in BEAS-2B cells. The in vivo study explored demoting effects of 10-20 mg/kg kaempferol on airway fibrosis in BALB/c mice sensitized with ovalbumin (OVA). LPS induced airway epithelial TGF-β1 signaling that promoted EMT with concurrent loss of E-cadherin and induction of α-smooth muscle actin (α-SMA). Nontoxic kaempferol significantly inhibited TGF-β-induced EMT process through reversing E-cadherin expression and retarding the induction of N-cadherin and α-SMA. Consistently, OVA inhalation resulted in a striking loss of epithelial morphology by displaying myofibroblast appearance, which led to bronchial fibrosis with submucosal accumulation of collagen fibers. Oral administration of kaempferol suppressed collagen deposition, epithelial excrescency and goblet hyperplasia observed in the lung of OVA-challenged mice. The specific inhibition of TGF-β entailed epithelial protease-activated receptor-1 (PAR-1) as with 20 μM kaempferol. The epithelial PAR-1 inhibition by SCH-79797 restored E-cadherin induction and deterred α-SMA induction, indicating that epithelial PAR-1 localization was responsible for resulting in airway EMT. These results demonstrate that dietary kaempferol alleviated fibrotic airway remodeling via bronchial EMT by modulating PAR1 activation. Therefore, kaempferol may be a potential therapeutic agent targeting asthmatic airway constriction.

Nicotine signals through muscle-type and neuronal nicotinic acetylcholine receptors in both human bronchial epithelial cells and airway fibroblasts

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Luketich James D

2004-12-01

Full Text Available Abstract Background Non-neuronal cells, including those derived from lung, are reported to express nicotinic acetylcholine receptors (nAChR. We examined nAChR subunit expression in short-term cultures of human airway cells derived from a series of never smokers, ex-smokers, and active smokers. Methods and Results At the mRNA level, human bronchial epithelial (HBE cells and airway fibroblasts expressed a range of nAChR subunits. In multiple cultures of both cell types, mRNA was detected for subunits that constitute functional muscle-type and neuronal-type pentomeric receptors. Two immortalized cell lines derived from HBE cells also expressed muscle-type and neuronal-type nAChR subunits. Airway fibroblasts expressed mRNA for three muscle-type subunits (α1, δ, and ε significantly more often than HBE cells. Immunoblotting of HBE cell and airway fibroblast extracts confirmed that mRNA for many nAChR subunits is translated into detectable levels of protein, and evidence of glycosylation of nAChRs was observed. Some minor differences in nAChR expression were found based on smoking status in fibroblasts or HBE cells. Nicotine triggered calcium influx in the immortalized HBE cell line BEAS2B, which was blocked by α-bungarotoxin and to a lesser extent by hexamethonium. Activation of PKC and MAPK p38, but not MAPK p42/44, was observed in BEAS2B cells exposed to nicotine. In contrast, nicotine could activate p42/44 in airway fibroblasts within five minutes of exposure. Conclusions These results suggest that muscle-type and neuronal-type nAChRs are functional in airway fibroblasts and HBE cells, that prior tobacco exposure does not appear to be an important variable in nAChR expression, and that distinct signaling pathways are observed in response to nicotine.

31P-nuclear magnetic resonance analysis of extracts of vascular smooth muscle

International Nuclear Information System (INIS)

Barron, J.T.; Messer, J.V.; Glonek, Thomas

1986-01-01

31 P-nuclear magnetic resonance spectroscopy was used to assess phosphate metabolites in perchloric acid extracts of rabbit aorta. In addition to the high energy phosphates, several other phosphorus compounds were detected and quantified. Most notable was the presence of a prominent phosphomonoester compound appearing at a chemical shift of 3.86 delta. This compound constituted 26% of the total extractable tissue phosphorus and is tentatively identified as ribose-5-phosphate, a pentose phosphate pathway intermediate. While ATP and phosphocreatine did not change during glucose and oxygen deprivation or during prolonged muscle contraction, the 3.86delta phosphate decreased significantly. Furthermore, theophylline, an agent that increases intracellular cAMP, also decreased the level of the 3.86 delta phosphate. These results are consistent with the concept that intermediate metabolism sustains high energy phosphate pools in vascular smooth muscle in the steady state under various conditions. The pentose phosphate pathway may play an important role in vascular smooth muscle metabolism. (author)

Myosin Va Plays a Role in Nitrergic Smooth Muscle Relaxation in Gastric Fundus and Corpora Cavernosa of Penis

Science.gov (United States)

Carew, Josephine A.; Goyal, Raj K.; Sullivan, Maryrose P.

2014-01-01

The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction. PMID:24516539

Bone Morphogenetic Protein-Induced Msx1 and Msx2 Inhibit Myocardin-Dependent Smooth Muscle Gene Transcription▿

OpenAIRE

Hayashi, Ken'ichiro; Nakamura, Seiji; Nishida, Wataru; Sobue, Kenji

2006-01-01

During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene ex...

Innate immune receptors in human airway smooth muscle cells: activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 agonists.

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Anne Månsson Kvarnhammar

Full Text Available BACKGROUND: Pattern-recognition receptors (PRRs, including Toll-like receptors (TLRs, NOD-like receptors (NLRs and RIG-I-like receptors (RLRs, recognize microbial components and trigger a host defense response. Respiratory tract infections are common causes of asthma exacerbations, suggesting a role for PRRs in this process. The present study aimed to examine the expression and function of PRRs on human airway smooth muscle cells (HASMCs. METHODS: Expression of TLR, NLR and RLR mRNA and proteins was determined using real-time RT-PCR, flow cytometry and immunocytochemistry. The functional responses to ligand stimulation were investigated in terms of cytokine and chemokine release, cell surface marker expression, proliferation and proteins regulating the contractile state. RESULTS: HASMCs expressed functional TLR2, TLR3, TLR4, TLR7 and NOD1. Stimulation with the corresponding agonists Pam3CSK4, poly(I:C, LPS, R-837 and iE-DAP, respectively, induced IL-6, IL-8 and GM-CSF release and up-regulation of ICAM-1 and HLA-DR, while poly(I:C also affected the release of eotaxin and RANTES. The proliferative response was slightly increased by LPS. Stimulation, most prominently with poly(I:C, down-regulated myosin light chain kinase and cysteinyl leukotriene 1 receptor expression and up-regulated β2-adrenoceptor expression. No effects were seen for agonist to TLR2/6, TLR5, TLR8, TLR9, NOD2 or RIG-I/MDA-5. CONCLUSION: Activation of TLR2, TLR3, TLR4, TLR7 and NOD1 favors a synthetic phenotype, characterized by an increased ability to release inflammatory mediators, acquire immunomodulatory properties by recruiting and interacting with other cells, and reduce the contractile state. The PRRs might therefore be of therapeutic use in the management of asthma and infection-induced disease exacerbations.

Rapid effects of phytoestrogens on human colonic smooth muscle are mediated by oestrogen receptor beta.

LENUS (Irish Health Repository)

Hogan, A M

2012-02-01

Epidemiological studies have correlated consumption of dietary phytoestrogens with beneficial effects on colon, breast and prostate cancers. Genomic and non-genomic mechanisms are responsible for anti-carcinogenic effects but, until now, the effect on human colon was assumed to be passive and remote. No direct effect on human colonic smooth muscle has previously been described. Institutional research board approval was granted. Histologically normal colon was obtained from the proximal resection margin of colorectal carcinoma specimens. Circular smooth muscle strips were microdissected and suspended under 1g of tension in organ baths containing oxygenated Krebs solution at 37 degrees C. After an equilibration period, tissues were exposed to diarylpropionitrile (DPN) (ER beta agonist) and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) (ER alpha agonist) or to the synthetic phytoestrogen compounds genistein (n=8), daidzein (n=8), fisetin (n=8) and quercetin (n=8) in the presence or absence of fulvestrant (oestrogen receptor antagonist). Mechanism of action was investigated by inhibition of downstream pathways. The cholinergic agonist carbachol was used to induce contractile activity. Tension was recorded isometrically. Phytoestrogens inhibit carbachol-induced colonic contractility. In keeping with a non-genomic, rapid onset direct action, the effect was within minutes, reversible and similar to previously described actions of 17 beta oestradiol. No effect was seen in the presence of fulvestrant indicating receptor modulation. While the DPN exerted inhibitory effects, PPT did not. The effect appears to be reliant on a p38\\/mitogen activated protein kinase mediated induction of nitric oxide production in colonic smooth muscle. The present data set provides the first description of a direct effect of genistein, daidzein, fisetin and quercetin on human colonic smooth muscle. The presence of ER in colonic smooth muscle has been functionally proven and the beta

Deficiency of RAMP1 attenuates antigen-induced airway hyperresponsiveness in mice.

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Manyu Li

Full Text Available Asthma is a chronic inflammatory disease affecting the lung, characterized by breathing difficulty during an attack following exposure to an environmental trigger. Calcitonin gene-related peptide (CGRP is a neuropeptide that may have a pathological role in asthma. The CGRP receptor is comprised of two components, which include the G-protein coupled receptor, calcitonin receptor-like receptor (CLR, and receptor activity-modifying protein 1 (RAMP1. RAMPs, including RAMP1, mediate ligand specificity in addition to aiding in the localization of receptors to the cell surface. Since there has been some controversy regarding the effect of CGRP on asthma, we sought to determine the effect of CGRP signaling ablation in an animal model of asthma. Using gene-targeting techniques, we generated mice deficient for RAMP1 by excising exon 3. After determining that these mice are viable and overtly normal, we sensitized the animals to ovalbumin prior to assessing airway resistance and inflammation after methacholine challenge. We found that mice lacking RAMP1 had reduced airway resistance and inflammation compared to wildtype animals. Additionally, we found that a 50% reduction of CLR, the G-protein receptor component of the CGRP receptor, also ameliorated airway resistance and inflammation in this model of allergic asthma. Interestingly, the loss of CLR from the smooth muscle cells did not alter the airway resistance, indicating that CGRP does not act directly on the smooth muscle cells to drive airway hyperresponsiveness. Together, these data indicate that signaling through RAMP1 and CLR plays a role in mediating asthma pathology. Since RAMP1 and CLR interact to form a receptor for CGRP, our data indicate that aberrant CGRP signaling, perhaps on lung endothelial and inflammatory cells, contributes to asthma pathophysiology. Finally, since RAMP-receptor interfaces are pharmacologically tractable, it may be possible to develop compounds targeting the RAMP1/CLR

Acute effect of insulin on guinea pig airways and its amelioration by pre-treatment with salbutamol

International Nuclear Information System (INIS)

Sharif, M.; Khan, B. T.; Anwar, M. A.

2014-01-01

Objective: To study the magnitude of insulin-mediated airway hyper-reactivity and to explore the protective effects of salbutamol in inhibiting the insulin-induced airway hyper-responsiveness on tracheal smooth muscle of guinea pigs in vitro. Methods: The quasi-experimental study was conducted at the Pharmacology Department of Army Medical College, Rawalpindi, in collaboration with the Centre for Research in Experimental and Applied Medicine from December 2011 to July 2012. It used 18 healthy Dunkin Hartely guinea pigs of either gender. Effects of increasing concentrations of histamine (10-8-10-3M), insulin (10-8-10-3 M) and insulin pre-treated with salbutamol (10-6 M) were observed on isolated tracheal strip of guinea pig in vitro by constructing cumulative concentration response curves. The tracheal smooth muscle contractions were recorded with Transducer on Four Channel Oscillograph. Mean and standard error of mean were calculated. SPSS 16 was used for statistical analysis. Results: Histamine and insulin produced a concentration-dependent reversible contraction of isolated tracheal muscle of guinea pig. The mean of maximum amplitudes of contraction with histamine, insulin and insulin pre-treated with salbutamol were 92. 1.20 mm, 35+-1.13 mm and 14.55+-0.62 mm respectively. Salbutamol shifted the concentration response curve of insulin to the right and downwards. Conclusions: Salbutamol significantly reduced the insulin mediated airway hyper-reactivity in guinea pigs, suggesting that pre-treatment of inhaled insulin with salbutamol may have clinical implication in the amelioration of its potential respiratory adverse effects such as bronchoconstriction. (author)

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

International Nuclear Information System (INIS)

Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

2011-01-01

Highlights: ► Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. ► Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. ► It is a widely believed that MYL2 is a poor substrate for smMLCK. ► In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. ► Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca 2+ sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis–Menten V max and K M for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

Energy Technology Data Exchange (ETDEWEB)

Josephson, Matthew P.; Sikkink, Laura A. [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Penheiter, Alan R. [Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905 (United States); Burghardt, Thomas P., E-mail: burghardt@mayo.edu [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905 (United States); Ajtai, Katalin [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)

2011-12-16

Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

Associations between airway hyperresponsiveness, obesity, and lipoproteins in a longitudinal cohort

DEFF Research Database (Denmark)

Rasmussen, Finn; Hancox, Robert; Nair, Parameswaran

2013-01-01

Background Epidemiological data have linked obesity with an increased risk of asthma, but the mechanisms responsible for this relationship remain unknown. One possible mechanism, which has received little attention, could be the effect of cholesterol metabolism on airway smooth muscle function. T...... assessed in a population-based cohort at ages 14 and 20 years. Results In unadjusted analyses, higher LDL cholesterol levels at age 14 were associated with AHR at age 20 in both sexes (P...

The force recovery following repeated quick releases applied to pig urinary bladder smooth muscle

NARCIS (Netherlands)

R. van Mastrigt (Ron)

1991-01-01

textabstractA method for measuring several quick-releases during one contraction of a pig urinary bladder smooth muscle preparation was developed. The force recovery following quick release in this muscle type was studied by fitting a multiexponential model to 926 responses measured during the first

Isolation of pulmonary artery smooth muscle cells from neonatal mice.

Science.gov (United States)

Lee, Keng Jin; Czech, Lyubov; Waypa, Gregory B; Farrow, Kathryn N

2013-10-19

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al. that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 μM in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

Chitosan-based scaffolds for the support of smooth muscle constructs in intestinal tissue engineering

Science.gov (United States)

Zakhem, Elie; Raghavan, Shreya; Gilmont, Robert R; Bitar, Khalil N

2012-01-01

Intestinal tissue engineering is an emerging field due to a growing demand for intestinal lengthening and replacement procedures secondary to massive resections of the bowel. Here, we demonstrate the potential use of a chitosan/collagen scaffold as a 3D matrix to support the bioengineered circular muscle constructs maintain their physiological functionality. We investigated the biocompatibility of chitosan by growing rabbit colonic circular smooth muscle cells (RCSMCs) on chitosan-coated plates. The cells maintained their spindle-like morphology and preserved their smooth muscle phenotypic markers. We manufactured tubular scaffolds with central openings composed of chitosan and collagen in a 1:1 ratio. Concentrically-aligned 3D circular muscle constructs were bioengineered using fibrin-based hydrogel seeded with RCSMCs. The constructs were placed around the scaffold for 2 weeks, after which they were taken off and tested for their physiological functionality. The muscle constructs contracted in response to Acetylcholine (Ach) and potassium chloride (KCl) and they relaxed in response to vasoactive intestinal peptide (VIP). These results demonstrate that chitosan is a biomaterial possibly suitable for intestinal tissue engineering applications. PMID:22483012

Heterogeneity of smooth muscle cells in tunica media of aorta in ...

African Journals Online (AJOL)

... of the tunica media of goat aorta are phenotypically heterogeneous and run in multiple directions. These characteristics probably confer mechanical strength and functional plasticity to the aortic wall. Designers of aortic substitutes should bear this in mind. Keywords: Vascular, Smooth Muscle Cells, Heterogeneity, Aorta ...

Akt1/PKB upregulation leads to vascular smooth muscle cell hypertrophy and polyploidization

Science.gov (United States)

Hixon, Mary L.; Muro-Cacho, Carlos; Wagner, Mark W.; Obejero-Paz, Carlos; Millie, Elise; Fujio, Yasushi; Kureishi, Yasuko; Hassold, Terry; Walsh, Kenneth; Gualberto, Antonio

2000-01-01

Vascular smooth muscle cells (VSMCs) at capacitance arteries of hypertensive individuals and animals undergo marked age- and blood pressure–dependent polyploidization and hypertrophy. We show here that VSMCs at capacitance arteries of rat models of hypertension display high levels of Akt1/PKB protein and activity. Gene transfer of Akt1 to VSMCs isolated from a normotensive rat strain was sufficient to abrogate the activity of the mitotic spindle cell–cycle checkpoint, promoting polyploidization and hypertrophy. Furthermore, the hypertrophic agent angiotensin II induced VSMC polyploidization in an Akt1-dependent manner. These results demonstrate that Akt1 regulates ploidy levels in VSMCs and contributes to vascular smooth muscle polyploidization and hypertrophy during hypertension. PMID:11032861

The Kinetics of Myosin Light Chain Kinase Activation of Smooth Muscle Myosin in an In Vitro Model System

OpenAIRE

Hong, Feng; Facemyer, Kevin C.; Carter, Michael S.; Jackson, Del R.; Haldeman, Brian D.; Ruana, Nick; Sutherland, Cindy; Walsh, Michael P.; Cremo, Christine R.; Baker, Josh E.

2013-01-01

During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca2+-CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vi...

Differential gene expression profiling of human adipose stem cells differentiating into smooth muscle-like cells by TGFβ1/BMP4

Energy Technology Data Exchange (ETDEWEB)

Elçin, Ayşe Eser; Parmaksiz, Mahmut; Dogan, Arin; Seker, Sukran; Durkut, Serap; Dalva, Klara; Elçin, Yaşar Murat, E-mail: elcinmurat@gmail.com

2017-03-15

Regenerative repair of the vascular system is challenging from the perspectives of translational medicine and tissue engineering. There are fundamental hurdles in front of creating bioartificial arteries, which involve recaputilation of the three-layered structure under laboratory settings. Obtaining and maintaining smooth muscle characteristics is an important limitation, as the transdifferentiated cells fail to display mature phenotype. This study aims to shed light on the smooth muscle differentiation of human adipose stem cells (hASCs). To this end, we first acquired hASCs from lipoaspirate samples. Upon characterization, the cells were induced to differentiate into smooth muscle (SM)-like cells using a variety of inducer combinations. Among all, TGFβ1/BMP4 combination had the highest differentiation efficiency, based on immunohistochemical analyses. hSM-like cell samples were compared to hASCs and to the positive control, human coronary artery-smooth muscle cells (hCA-SMCs) through gene transcription profiling. Microarray findings revealed the activation of gene groups that function in smooth muscle differentiation, signaling pathways, extracellular modeling and cell proliferation. Our results underline the effectiveness of the growth factors and suggest some potential variables for detecting the SM-like cell characteristics. Evidence in transcriptome level was used to evaluate the TGFβ1/BMP4 combination as a previously unexplored effector for the smooth muscle differentiation of adipose stem cells. - Highlights: • Human adipose stem cells (hASCs) were isolated, characterized and cultured. • Growth factor combinations were evaluated for their effectiveness in differentiation using IHC. • hASCs were differentiated into smooth muscle (SM)-like cells using TGF-β1 and BMP4 combination. • Microarray analysis was performed for hASCs, SM-like cells and coronary artery-SMCs. • Microarray data was used to perform hierarchical clustering and interpretation

Oxygen mediates vascular smooth muscle relaxation in hypoxia.

Directory of Open Access Journals (Sweden)

Jessica Dada

Full Text Available The activation of soluble guanylate cyclase (sGC by nitric oxide (NO and other ligands has been extensively investigated for many years. In the present study we considered the effect of molecular oxygen (O2 on sGC both as a direct ligand and its affect on other ligands by measuring cyclic guanosine monophosphate (cGMP production, as an index of activity, as well as investigating smooth muscle relaxation under hypoxic conditions. Our isolated enzyme studies confirm the function of sGC is impaired under hypoxic conditions and produces cGMP in the presence of O2, importantly in the absence of NO. We also show that while O2 could partially affect the magnitude of sGC stimulation by NO when the latter was present in excess, activation by the NO independent, haem-dependent sGC stimulator 3-(5'-hydroxymethyl-2'-furyl-1-benzylindazole (YC-1 was unaffected. Our in vitro investigation of smooth muscle relaxation confirmed that O2 alone in the form of a buffer bolus (equilibrated at 95% O2/5% CO2 had the ability to dilate vessels under hypoxic conditions and that this was dependent upon sGC and independent of eNOS. Our studies confirm that O2 can be a direct and important mediator of vasodilation through an increase in cGMP production. In the wider context, these observations are key to understanding the relative roles of O2 versus NO-induced sGC activation.

Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome.

Science.gov (United States)

Crosas-Molist, Eva; Meirelles, Thayna; López-Luque, Judit; Serra-Peinado, Carla; Selva, Javier; Caja, Laia; Gorbenko Del Blanco, Darya; Uriarte, Juan José; Bertran, Esther; Mendizábal, Yolanda; Hernández, Vanessa; García-Calero, Carolina; Busnadiego, Oscar; Condom, Enric; Toral, David; Castellà, Manel; Forteza, Alberto; Navajas, Daniel; Sarri, Elisabet; Rodríguez-Pascual, Fernando; Dietz, Harry C; Fabregat, Isabel; Egea, Gustavo

2015-04-01

Marfan's syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-β signaling. TGF-β is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-β signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan's syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-β pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. In Marfan VSMC, both in tissue and in culture, there are variable TGF-β-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan's syndrome aneurysm formation. © 2015 American Heart Association, Inc.

Relaxant effect of ghrelin on guinea pig isolated tracheal smooth muscle: role of epithelial NO and PGE2.

Science.gov (United States)

Al-Ayed, Mohammed Saeed Zayed

2018-06-01

This study aimed at investigating the potential ghrelin relaxing effect on guinea pig isolated tracheal smooth muscle (TSM). Using an in vitro experimental approach, the physiological role of the airway epithelium on smooth muscle relaxation has been investigated by analyzing the dose-response curves for carbachol- or histamine-induced contractions on epithelium intact versus denuded tracheal tissue. The relaxant effect of ghrelin (5-200 μmol/L) then investigated on carbachol-contracted, non-sensitized, and ovalbumin (OVA)-sensitized guinea pig TSM with an intact or denuded epithelium. The isolated TSMs from identical guinea pigs were incubated in Krebs solution aerated with 95% O 2 and 5% CO 2 through an automated tissue organ bath system (n = 6 for each group). The ghrelin relaxation mechanism was assessed by adding L-NAME, indomethacin, and YIL-781 for GHS-R1 into the tissue chamber. The spasmogens carbachol and histamine have shown a significantly higher contracting effect on epithelium-denuded than in epithelium-intact TSM confirmed by the significantly higher mean pEC50 of both agonists on the epithelium-denuded trachea (p < 0.05). Ghrelin has shown a concentration-dependent relaxing effect on carbachol-contracted TSM (r = 0.96, p = 0.00). The effect was more evident in the intact non-sensitized than in epithelium-denuded or OVA-sensitized groups (p < 0.05). Preincubation with nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) inhibitors has significantly reduced the ghrelin-induced relaxation on epithelium-intact TSM suggesting an epithelium-dependant mechanism. However, GHS-R1a antagonist has also succeeded to reduce ghrelin relaxant effect, which needs further clarification. Ghrelin proved to have a potential TSM relaxant effect possibly through epithelium-dependant mechanisms involving NO and PGE 2 .

Role of cyclic GMP in cells with the properties of smooth muscle cultured from the rat myometrium

International Nuclear Information System (INIS)

Krall, J.F.; Morin, A.

1986-01-01

Cells growing in culture with previously described properties of rat uterine smooth muscle accumulated 45 Ca 2+ from the medium. Ca 2+ uptake by these cells was stimulated by the addition to the medium of 8-bromo-cGMP but not by 8-bromo-cAMP. Ca 2+ uptake was also stimulated by carbachol and by the nitro-vasodilator nitroprusside. Although cholinergic agonists have been shown previously to stimulate contraction but not cGMP synthesis in the rat myometrium, both carbachol and nitroprusside stimulated cGMP production by the cultured cells. These results suggested the cells had cholinergic receptor-medicated functions that reflected some neurotransmitter-sensitive properties of uterine smooth muscle in situ. When determined by a specific radioligand binding assay, subcellular fractions of the cultured cells bound muscarinic cholinergic agonists and antagonists with affinities expected of the muscarinic receptor. The cells were also sensitive to the β-adrenergic catecholamine agonist isoproterenol, which stimulated cAMP production but not Ca 2+ uptake. Carbachol failed to inhibit isoproterenol-dependent cAMP production, which is an important property of the cholinergic receptor in uterine smooth muscle in situ. These results suggest some but not all acetylcholine-sensitive properties of uterine smooth muscle may be retained in cell culture

Macrophage secretory products selectively stimulate dermatan sulfate proteoglycan production in cultured arterial smooth muscle cells

International Nuclear Information System (INIS)

Edwards, I.J.; Wagner, W.D.; Owens, R.T.

1990-01-01

Arterial dermatan sulfate proteoglycan has been shown to increase with atherosclerosis progression, but factors responsible for this increase are unknown. To test the hypothesis that smooth muscle cell proteoglycan synthesis may be modified by macrophage products, pigeon arterial smooth muscle cells were exposed to the media of either cholesteryl ester-loaded pigeon peritoneal macrophages or a macrophage cell line P388D1. Proteoglycans radiolabeled with [35S]sulfate and [3H]serine were isolated from culture media and smooth muscle cells and purified following precipitation with 1-hexadecylpyridinium chloride and chromatography. Increasing concentrations of macrophage-conditioned media were associated with a dose-response increase in [35S]sulfate incorporation into secreted proteoglycans, but there was no change in cell-associated proteoglycans. Incorporation of [3H]serine into total proteoglycan core proteins was not significantly different (5.2 X 10(5) dpm and 5.5 X 10(5) disintegrations per minute (dpm) in control and conditioned media-treated cultures, respectively), but selective effects were observed on individual proteoglycan types. Twofold increases in dermatan sulfate proteoglycan and limited degradation of chondroitin sulfate proteoglycan were apparent based on core proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoinhibition studies indicated that interleukin-1 was involved in the modulation of proteoglycan synthesis by macrophage-conditioned media. These data provide support for the role of macrophages in alteration of the matrix proteoglycans synthesized by smooth muscle cells and provide a mechanism to account for the reported increased dermatan sulfate/chondroitin sulfate ratios in the developing atherosclerotic lesion

Calcification of human vascular smooth muscle cells: associations with osteoprotegerin expression and acceleration by high-dose insulin

DEFF Research Database (Denmark)

Olesen, Ping; Knudsen, Kirsten Quyen Nguyen; Wogensen, Lise

2007-01-01

Arterial medial calcifications occur often in diabetic individuals as part of the diabetic macroangiopathy. The pathogenesis is unknown, but the presence of calcifications predicts risk of cardiovascular events. We examined the effects of insulin on calcifying smooth muscle cells in vitro...... and measured the expression of the bone-related molecule osteoprotegerin (OPG). Human vascular smooth muscle cells (VSMCs) were grown from aorta from kidney donors. Induction of calcification was performed with beta-glycerophosphate. The influence of insulin (200 microU/ml or 1,000 microU/ml) on calcification...... calcification in human smooth muscle cells from a series of donors after variable time in culture. Decreased OPG amounts were observed from the cells during the accelerated calcification phase. High dose of insulin (1,000 microU/ml) accelerated the calcification, whereas lower concentrations (200 microU/ml) did...

Ionotropic and Metabotropic Proton-Sensing Receptors Involved in Airway Inflammation in Allergic Asthma

Directory of Open Access Journals (Sweden)

Haruka Aoki

2014-01-01

Full Text Available An acidic microenvironment has been shown to evoke a variety of airway responses, including cough, bronchoconstriction, airway hyperresponsiveness (AHR, infiltration of inflammatory cells in the lung, and stimulation of mucus hyperproduction. Except for the participation of transient receptor potential vanilloid-1 (TRPV1 and acid-sensing ion channels (ASICs in severe acidic pH (of less than 6.0-induced cough and bronchoconstriction through sensory neurons, the molecular mechanisms underlying extracellular acidic pH-induced actions in the airways have not been fully understood. Recent studies have revealed that ovarian cancer G protein-coupled receptor 1 (OGR1-family G protein-coupled receptors, which sense pH of more than 6.0, are expressed in structural cells, such as airway smooth muscle cells and epithelial cells, and in inflammatory and immune cells, such as eosinophils and dendritic cells. They function in a variety of airway responses related to the pathophysiology of inflammatory diseases, including allergic asthma. In the present review, we discuss the roles of ionotropic TRPV1 and ASICs and metabotropic OGR1-family G protein-coupled receptors in the airway inflammation and AHR in asthma and respiratory diseases.

A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

DEFF Research Database (Denmark)

Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

1992-01-01

major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10......Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...

Individual sympathetic postganglionic neurons coinnervate myenteric ganglia and smooth muscle layers in the gastrointestinal tract of the rat.

Science.gov (United States)

Walter, Gary C; Phillips, Robert J; McAdams, Jennifer L; Powley, Terry L

2016-09-01

A full description of the terminal architecture of sympathetic axons innervating the gastrointestinal (GI) tract has not been available. To label sympathetic fibers projecting to the gut muscle wall, dextran biotin was injected into the celiac and superior mesenteric ganglia (CSMG) of rats. Nine days postinjection, animals were euthanized and stomachs and small intestines were processed as whole mounts (submucosa and mucosa removed) to examine CSMG efferent terminals. Myenteric neurons were counterstained with Cuprolinic Blue; catecholaminergic axons were stained immunohistochemically for tyrosine hydroxylase. Essentially all dextran-labeled axons (135 of 136 sampled) were tyrosine hydroxylase-positive. Complete postganglionic arbors (n = 154) in the muscle wall were digitized and analyzed morphometrically. Individual sympathetic axons formed complex arbors of varicose neurites within myenteric ganglia/primary plexus and, concomitantly, long rectilinear arrays of neurites within circular muscle/secondary plexus or longitudinal muscle/tertiary plexus. Very few CSMG neurons projected exclusively (i.e., ∼100% of an arbor's varicose branches) to myenteric plexus (∼2%) or smooth muscle (∼14%). With less stringent inclusion criteria (i.e., ≥85% of an axon's varicose branches), larger minorities of neurons projected predominantly to either myenteric plexus (∼13%) or smooth muscle (∼27%). The majority (i.e., ∼60%) of all individual CSMG postganglionics formed mixed, heterotypic arbors that coinnervated extensively (>15% of their varicose branches per target) both myenteric ganglia and smooth muscle. The fact that ∼87% of all sympathetics projected either extensively or even predominantly to smooth muscle, while simultaneously contacting myenteric plexus, is consistent with the view that these neurons control GI muscle directly, if not exclusively. J. Comp. Neurol. 524:2577-2603, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Microtubule Regulation of Kv7 Channels Orchestrates cAMP-Mediated Vasorelaxations in Rat Arterial Smooth Muscle

DEFF Research Database (Denmark)

Lindman, Johanna; Khammy, Makhala M; Lundegaard, Pia R

2018-01-01

Microtubules can regulate GPCR (G protein-coupled receptor) signaling in various cell types. In vascular smooth muscle, activation of the β-adrenoceptor leads to production of cAMP to mediate a vasorelaxation. Little is known about the role of microtubules in smooth muscle, and given the importance...... of renal and mesenteric arteries that the microtubule stabilizer, paclitaxel, prevented. Sharp microelectrode experiments showed that colchicine treatment caused increased hyperpolarization of mesenteric artery segments in response to isoprenaline. Application of the Kv7 channel blocker, XE991, attenuated...

The length dependence of the series elasticity of pig bladder smooth muscle

NARCIS (Netherlands)

R. van Mastrigt (Ron)

1988-01-01

textabstractStrips of urinary bladder smooth muscle were subjected to a series of quick release measurements. Each measurement consisted of several releases and resets to the original length, made during one contraction. The complete length-force characteristic of series elasticity was quantified by

Preliminary investigations on the effects of a Strongylus vulgaris larval extract, mononuclear factors and platelet factors on equine smooth muscle cells in vitro.

Science.gov (United States)

Morgan, S J; Storts, R W; Stromberg, P C; Sowa, B A; Lay, J C

1989-01-01

Factors involved in the proliferation of equine vascular smooth muscle cells were studied in vitro. The most prominent proliferative responses in cultured vascular smooth muscle cells were induced by Strongylus vulgaris larval antigen extract (LAE) and platelet-derived factors. Less significant proliferative responses were obtained with conditioned media from S. vulgaris LAE stimulated and from unstimulated equine mononuclear leukocytes. Additionally, vascular smooth muscle cells exposed to S. vulgaris LAE developed numerous perinuclear vacuoles and were more spindle-shaped than control or smooth muscle cells exposed to other factors. Equine mononuclear leukocytes exposed to LAE developed prominent morphological changes, including enlargement, clumping and increased numbers of mitotic figures.

Possible role of differential growth in airway wall remodeling in asthma

KAUST Repository

Moulton, D. E.

2011-01-20

Possible role of differential growth in airway wall remodeling in asthma. J Appl Physiol 110: 1003-1012, 2011. First published January 20, 2011; doi:10.1152/japplphysiol.00991.2010.- Airway remodeling in patients with chronic asthma is characterized by a thickening of the airway walls. It has been demonstrated in previous theoretical models that this change in thickness can have an important mechanical effect on the properties of the wall, in particular on the phenomenon of mucosal folding induced by smooth muscle contraction. In this paper, we present a model for mucosal folding of the airway in the context of growth. The airway is modeled as a bilayered cylindrical tube, with both geometric and material nonlinearities accounted for via the theory of finite elasticity. Growth is incorporated into the model through the theory of morphoelasticity. We explore a range of growth possibilities, allowing for anisotropic growth as well as different growth rates in each layer. Such nonuniform growth, referred to as differential growth, can change the properties of the material beyond geometrical changes through the generation of residual stresses. We demonstrate that differential growth can have a dramatic impact on mucosal folding, in particular on the critical pressure needed to induce folding, the buckling pattern, as well as airway narrowing. We conclude that growth may be an important component in airway remodeling. Copyright © 2011 the American Physiological Society.

Beneficial effects of ursodeoxycholic acid via inhibition of airway remodelling, apoptosis of airway epithelial cells, and Th2 immune response in murine model of chronic asthma.

Science.gov (United States)

Işık, S; Karaman, M; Çilaker Micili, S; Çağlayan-Sözmen, Ş; Bağrıyanık, H Alper; Arıkan-Ayyıldız, Z; Uzuner, N; Karaman, Ö

In previous studies, anti-inflammatory, anti-apoptotic and immunomodulatory effects of ursodeoxycholic acid (UDCA) on liver diseases have been shown. In this study, we aimed to investigate the effects of UDCA on airway remodelling, epithelial apoptosis, and T Helper (Th)-2 derived cytokine levels in a murine model of chronic asthma. Twenty-seven BALB/c mice were divided into five groups; PBS-Control, OVA-Placebo, OVA-50mg/kg UDCA, OVA-150mg/kg UDCA, OVA-Dexamethasone. Mice in groups OVA-50mg/kg UDCA, OVA-150mg/kg UDCA, OVA-Dexamethasone received the UDCA (50mg/kg), UDCA (150mg/kg), and dexamethasone, respectively. Epithelium thickness, sub-epithelial smooth muscle thickness, number of mast and goblet cells of samples isolated from the lung were measured. Immunohistochemical scorings of the lung tissue for matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEG-F), transforming growth factor-beta (TGF-β), terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) and cysteine-dependent aspartate-specific proteases (caspase)-3 were determined. IL-4, IL-5, IL-13, Nitric oxide, ovalbumin-specific immunoglobulin (Ig) E levels were quantified. The dose of 150mg/kg UDCA treatment led to lower epithelial thickness, sub-epithelial smooth muscle thickness, goblet and mast cell numbers compared to placebo. Except for MMP-9 and TUNEL all immunohistochemical scores were similar in both UDCA treated groups and the placebo. All cytokine levels were significantly lower in group IV compared to the placebo. These findings suggested that the dose of 150mg/kg UDCA improved all histopathological changes of airway remodelling and its beneficial effects might be related to modulating Th-2 derived cytokines and the inhibition of apoptosis of airway epithelial cells. Copyright © 2017 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.

Hepatocyte growth factor triggers signaling cascades mediating vascular smooth muscle cell migration

NARCIS (Netherlands)

Taher, Taher E. I.; Derksen, Patrick W. B.; de Boer, Onno J.; Spaargaren, Marcel; Teeling, Peter; van der Wal, Allard C.; Pals, Steven T.

2002-01-01

A key event in neointima formation and atherogenesis is the migration of vascular smooth muscle cells (VSMCs) into the intima. This is controlled by cytokines and extracellular matix (ECM) components within the microenvironment of the diseased vessel wall. At present, these signals have only been

Isolation and individual electrical stimulation of single smooth-muscle cells from the urinary bladder of the pig

NARCIS (Netherlands)

J.J. Glerum (Jacobus); R. van Mastrigt (Ron); J.C. Romijn (Johannes); D.J. Griffiths (Derek)

1987-01-01

textabstractIn contrast to striated muscle, measurements on strips of smooth muscle cannot be uniquely interpreted in terms of an array of contractile units. Therefore scaling down to the single-cell level is necessary to gain detailed understanding of the contractile process in this type of muscle.

TREK-1 Channel Expression in Smooth Muscle as a Target for Regulating Murine Intestinal Contractility: Therapeutic Implications for Motility Disorders

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Ruolin Ma

2018-03-01

Full Text Available Gastrointestinal (GI motility disorders such as irritable bowel syndrome (IBS can occur when coordinated smooth muscle contractility is disrupted. Potassium (K+ channels regulate GI smooth muscle tone and are key to GI tract relaxation, but their molecular and functional phenotypes are poorly described. Here we define the expression and functional roles of mechano-gated K2P channels in mouse ileum and colon. Expression and distribution of the K2P channel family were investigated using quantitative RT-PCR (qPCR, immunohistochemistry and confocal microscopy. The contribution of mechano-gated K2P channels to mouse intestinal muscle tension was studied pharmacologically using organ bath. Multiple K2P gene transcripts were detected in mouse ileum and colon whole tissue preparations. Immunohistochemistry confirmed TREK-1 expression was smooth muscle specific in both ileum and colon, whereas TREK-2 and TRAAK channels were detected in enteric neurons but not smooth muscle. In organ bath, mechano-gated K2P channel activators (Riluzole, BL-1249, flufenamic acid, and cinnamyl 1-3,4-dihydroxy-alpha-cyanocinnamate induced relaxation of KCl and CCh pre-contracted ileum and colon tissues and reduced the amplitude of spontaneous contractions. These data reveal the specific expression of mechano-gated K2P channels in mouse ileum and colon tissues and highlight TREK-1, a smooth muscle specific K2P channel in GI tract, as a potential therapeutic target for combating motility pathologies arising from hyper-contractility.

Evidence Supports Tradition: The in Vitro Effects of Roman Chamomile on Smooth Muscles

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Zsolt Sándor; Javad Mottaghipisheh; Katalin Veres; Judit Hohmann; Tímea Bencsik; Attila Horváth; Dezső Kelemen; Róbert Papp; Loránd Barthó; Dezső Csupor; Dezső Csupor

2018-01-01

The dried flowers of Chamaemelum nobile (L.) All. have been used in traditional medicine for different conditions related to the spasm of the gastrointestinal system. However, there have been no experimental studies to support the smooth muscle relaxant effect of this plant. The aim of our research was to assess the effects of the hydroethanolic extract of Roman chamomile, its fractions, four of its flavonoids (apigenin, luteolin, hispidulin, and eupafolin), and its essential oil on smooth mu...

Oxygenation decreases elastin secretion from rat ductus arteriosus smooth muscle cells.

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Kawakami, Shoji; Minamisawa, Susumu

2015-08-01

The ductus arteriosus (DA), a fetal arterial connection between the main pulmonary artery and the descending aorta, normally closes immediately after birth. The oxygen concentration in the blood rises after birth, and in the DA this increase in oxygen concentration causes functional closure, which is induced by smooth muscle contraction. Previous studies have demonstrated that hypoxia and/or oxygenation affect vascular remodeling of various vessels. Therefore, we hypothesized that the rise in oxygen concentration would affect the vascular structure of the DA due to production of proteins secreted from DA smooth muscle cells (SMC). Liquid chromatography-tandem mass spectrometry was used to comprehensively investigate the secreted proteins in the supernatant of rat DA SMC harvested under hypoxic conditions (1% oxygen) or under normoxic conditions (21% oxygen). We found that the rise in oxygen concentration reduced the secretion of elastin from DA SMC. On reverse transcription-polymerase chain reaction, the expression of elastin mRNA was not significantly changed in DA SMC from hypoxic to normoxic conditions. Given that elastin forms internal elastic lamina and elastic fibers in the vascular muscle layers, and that a rise in oxygen concentration reduced the secretion of elastin, this suggests that the rise in blood oxygen concentration after birth reduces the secretion of elastin, and therefore may play a role in DA structural remodeling after birth. © 2015 Japan Pediatric Society.

Hydro-ethanolic Extract of Portulaca oleracea Affects Beta-adrenoceptors of Guinea Pig Tracheal Smooth Muscle

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Boskabady, Mohammad Hossein; Hashemzehi, Milad; Khazdair, Mohammad Reza; Askari, Vahid Reza

2016-01-01

Thestimulatory effect of the extract of Portulaca oleracea (P. olerace) on β-adrenoceptor of tracheal smooth muscle was examined.To examine β-adrenoceptor stimulatory effect, concentration response curve to isoprenaline was obtained in pre-contracted tracheal smooth muscle in the presence of three concentrations of aqueous-ethanolic extract, propranolol, and saline. Values of EC50 (the effective concentration of isoprenaline, causing 50% of maximum response) and dose ratio-1(CR-1) were measured. This effect was tested innon-incubated tracheal smooth muscle (group 1) and incubated tissues with chlorpheniramine (group 2). Concentration-response curves to isoprenaline in the presence of two higher concentrations of the extract in group 1 and all three concentrations in group 2 showed leftward shifts compared to isoprenaline curves produced in the presence of saline in both groups. EC50 obtained in the presence of propranolol was significantly higher than that of saline in both groups of experiments (p<0.05 for both cases). However, the EC50 obtained in the presence of two higher concentrations of the extract in group 1 and lower concentration in group 2 were non-significantly but those obtained of medium and high extract concentrations in the group 2 were significantly (p<0.05 for both cases)lower than those of saline. The values of (CR-1) obtained in the presence of all concentrations of the extract in groups1 and 2 were significantly lower than that of propranolol (p<0.05 to p<0.001).The results indicated a stimulatory effect of the P. olerace extract on ß 2-adrenoceptors of tracheal smooth muscle. PMID:28243284

Smooth muscle LDL receptor-related protein-1 deletion induces aortic insufficiency and promotes vascular cardiomyopathy in mice.

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Joshua E Basford

Full Text Available Valvular disease is common in patients with Marfan syndrome and can lead to cardiomyopathy. However, some patients develop cardiomyopathy in the absence of hemodynamically significant valve dysfunction, suggesting alternative mechanisms of disease progression. Disruption of LDL receptor-related protein-1 (Lrp1 in smooth muscle cells has been shown to cause vascular pathologies similar to Marfan syndrome, with activation of smooth muscle cells, vascular dysfunction and aortic aneurysms. This study used echocardiography and blood pressure monitoring in mouse models to determine whether inactivation of Lrp1 in vascular smooth muscle leads to cardiomyopathy, and if so, whether the mechanism is a consequence of valvular disease. Hemodynamic changes during treatment with captopril were also assessed. Dilation of aortic roots was observed in young Lrp1-knockout mice and progressed as they aged, whereas no significant aortic dilation was detected in wild type littermates. Diastolic blood pressure was lower and pulse pressure higher in Lrp1-knockout mice, which was normalized by treatment with captopril. Aortic dilation was followed by development of aortic insufficiency and subsequent dilated cardiomyopathy due to valvular disease. Thus, smooth muscle cell Lrp1 deficiency results in aortic dilation and insufficiency that causes secondary cardiomyopathy that can be improved by captopril. These findings provide novel insights into mechanisms of cardiomyopathy associated with vascular activation and offer a new model of valvular cardiomyopathy.

Proliferation studies of the endothelial and smooth muscle cells of the mouse mesentery after irradiation

International Nuclear Information System (INIS)

Hirst, D.G.; Denekamp, J.; Hobson, B.

1980-01-01

A continuous tritium labelling technique was employed to study the effects of external β-radiation on the proliferation of endothelial cells and smooth muscle cells in the mesenteric arterioles of mice. Calculations showed very long turnover times for the two cell populations in control animals (> 2 years for endothelium and > 3 years for smooth muscle). After single doses of 20 and 45 Gy, no significant increase in endothelial proliferation was seen except at 3 weeks. No significant increase in labelling was observed in smooth muscle up to 48 weeks after irradiation. These labelling data have been compared with the pattern of cell depletion of the irradiated endothelium. It was concluded that the depletion was much earlier than expected for a slowly proliferating tissue, if all the cells were cycling very slowly. Such an early depletion is, however, consistent with cell death resulting from a small proportion of the cells having a short cell cycle. The recovery of the endothelial cell numbers between 9 and 12 months was not accompanied by a rise in the fraction of labelled cells. It is suggested that repopulation may occur from outside the treated area. (author)

Extracellular matrix of smooth muscle cells: interaction of collagen type V with heparan sulfate proteoglycan

International Nuclear Information System (INIS)

Gay, S.; Hoeoek, M.; Gay, R.E.; Magargal, W.W.; Reynertson, R.H.

1986-01-01

Alteration in the extracellular matrix produced by smooth muscle cells may play a role in the development of atherosclerotic lesions. Consequently the authors have initiated studies on the structural organization of the extracellular matrix produced by cultured smooth muscle cells. Immunohisotological examination of this matrix using well-characterized mono- and polyclonal antibodies showed a partial codistribution of heparan sulfate (HS) proteoglycans with a number of different matrix components including collagen types I, III, IV, V and VI, laminin and fibronectin. Subsequent binding studies between isolated matrix proteins and HS showed that the polysaccharide interacts strongly with type V collagen and to a lesser extent with fibronectin as well as collagen types III and VI. The interaction between type V and HS was readily inhibited by heparin and highly sulfated HS but not be dermatan sulfate, chondroitin sulfate or HS with a low sulfate content. Furthermore, [ 35 S]-HS proteoglycans isolated from cultured smooth muscle cells could be adsorbed on a column of sepharose conjugated with native type V collagen and eluted in a salt gradient. Hence, the interaction between type V and HS may play a major part in stabilizing the extracellular matrix of the vessel wall

[3H]QNB binding and contraction of rabbit colonic smooth muscle cells

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Ringer, M.J.; Hyman, P.E.; Kao, H.W.; Hsu, C.T.; Tomomasa, T.; Snape, W.J. Jr.

1987-01-01

The authors used radioligand binding and studies of cell contraction to characterize muscarinic receptors on dispersed smooth muscle cells from rabbit proximal and distal colon. Cells obtained after serial incubations in collagenase were used to measure binding of tritiated quinuclidinyl benzilate ([ 3 H]QNB). At 37 degree C, specific [ 3 H]QNB binding was saturable and linearly related to cell number. Nonlinear regression analysis was used to determine the affinity of [ 3 H]QNB for its receptor. The IC 50 for the muscarinic agonists bethanechol and oxotremorine were 80 and 0.57 μM, respectively. Hill coefficients were 0.67 for both, suggesting more complex interaction involving receptors of different affinities. In studies of cell contraction, bethanechol stimulated a dose-dependent decrease in cell length with half the maximal contraction occurring at 100 pM. These results suggest that (1) contraction is mediated by binding of bethanechol to M 2 -muscarinic receptors and that (2) there are a large number of spare receptors in colonic smooth muscle

Pulmonary Metastases of a Uterine Smooth Muscle Tumour with Undefined Malignancy Potential.

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Esch, M; Teschner, M; Braesen, J-H

2014-03-01

Smooth muscle neoplasms with atypical proliferative behaviour, but without clear histopathological malignancy represent a diagnostic and therapeutic challenge, as distinction from a sarcoma can be difficult and no guaranteed treatment recommendations are available due to the rarity of these changes. In the event of uncertain primary histology, even metastases cannot be assessed as malignancy criteria, but may contribute to the clarification of the histology. Similarities with other smooth muscle proliferations, such as lymphangioleiomyomatosis, are striking. The diagnostic difficulties and treatment options are explained based on the example of a 59-year-old patient, in whom a retroperitoneal mass and pulmonary lesion of such a tumour occurred 4 years after a hysterectomy. Even though the genesis and histological diagnostics have not been conclusively clarified, slow growth and a low recurrence rate for post-menopausal patients allow for a wait-and-see approach, whereby the option for anti-hormonal treatment exists in the event of positive evidence of hormone receptors.

Inflammation induced by mast cell deficiency rather than the loss of interstitial cells of Cajal causes smooth muscle dysfunction in W/Wv mice

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Winston, John H.; Chen, Jinghong; Shi, Xuan-Zheng; Sarna, Sushil K.

2014-01-01

The initial hypothesis suggested that the interstitial cells of Cajal (ICC) played an essential role in mediating enteric neuronal input to smooth muscle cells. Much information for this hypothesis came from studies in W/Wv mice lacking ICC. However, mast cells, which play critical roles in regulating inflammation in their microenvironment, are also absent in W/Wv mice. We tested the hypothesis that the depletion of mast cells in W/Wv mice generates inflammation in fundus muscularis externa (ME) that impairs smooth muscle reactivity to Ach, independent of the depletion of ICC. We performed experiments on the fundus ME from wild type (WT) and W/Wv mice before and after reconstitution of mast cells by bone marrow transplant. We found that mast cell deficiency in W/Wv mice significantly increased COX-2 and iNOS expression and decreased smooth muscle reactivity to Ach. Mast cell reconstitution or concurrent blockade of COX-2 and iNOS restored smooth muscle contractility without affecting the suppression of c-kit in W/Wv mice. The expression of nNOS and ChAT were suppressed in W/Wv mice; mast cell reconstitution did not restore them. We conclude that innate inflammation induced by mast cell deficiency in W/Wv mice impairs smooth muscle contractility independent of ICC deficiency. The impairment of smooth muscle contractility and the suppression of the enzymes regulating the synthesis of Ach and NO in W/Wv mice need to be considered in evaluating the role of ICC in regulating smooth muscle and enteric neuronal function in W/Wv mice. PMID:24550836

Estrogen-Induced Maldevelopment of the Penis Involves Down-Regulation of Myosin Heavy Chain 11 (MYH11) Expression, a Biomarker for Smooth Muscle Cell Differentiation1

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Okumu, L.A.; Bruinton, Sequoia; Braden, Tim D.; Simon, Liz; Goyal, Hari O.

2012-01-01

ABSTRACT Cavernous smooth muscle cells are essential components in penile erection. In this study, we investigated effects of estrogen exposure on biomarkers for smooth muscle cell differentiation in the penis. Neonatal rats received diethylstilbestrol (DES), with or without the estrogen receptor (ESR) antagonist ICI 182,780 (ICI) or the androgen receptor (AR) agonist dihydrotestosterone (DHT), from Postnatal Days 1 to 6. Tissues were collected at 7, 10, or 21 days of age. The smooth muscle cell biomarker MYH11 was studied in depth because microarray data showed it was significantly down-regulated, along with other biomarkers, in DES treatment. Quantitative real time-PCR and Western blot analyses showed 50%–80% reduction (P ≤ 0.05) in Myh11 expression in DES-treated rats compared to that in controls; and ICI and DHT coadministration mitigated the decrease. Temporally, from 7 to 21 days of age, Myh11 expression was onefold increased (P ≥ 0.05) in DES-treated rats versus threefold increased (P ≤ 0.001) in controls, implying the long-lasting inhibitory effect of DES on smooth muscle cell differentiation. Immunohistochemical localization of smooth muscle alpha actin, another biomarker for smooth muscle cell differentiation, showed fewer cavernous smooth muscle cells in DES-treated animals than in controls. Additionally, DES treatment significantly up-regulated Esr1 mRNA expression and suppressed the neonatal testosterone surge by 90%, which was mitigated by ICI coadministration but not by DHT coadministration. Collectively, results provided evidence that DES treatment in neonatal rats inhibited cavernous smooth muscle cell differentiation, as shown by down-regulation of MYH11 expression at the mRNA and protein levels and by reduced immunohistochemical staining of smooth muscle alpha actin. Both the ESR and the AR pathways probably mediate this effect. PMID:22976277

In vitro effects of oxytocin, acepromazine, detomidine, xylazine, butorphanol, terbutaline, isoproterenol, and dantrolene on smooth and skeletal muscles of the equine esophagus.

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Wooldridge, Anne A; Eades, Susan C; Hosgood, Giselle L; Moore, Rustin M

2002-12-01

To characterize the in vitro effects of oxytocin, acepromazine, xylazine, butorphanol, detomidine, dantrolene, isoproterenol, and terbutaline on skeletal and smooth muscle from the equine esophagus. 14 adult horses without digestive tract disease. Circular and longitudinal strips from the skeletal and smooth muscle of the esophagus were suspended in tissue baths, connected to force-displacement transducers interfaced with a physiograph, and electrical field stimulation was applied. Cumulative concentration-response curves were generated for oxytocin, acepromazine, xylazine, detomidine, butorphanol, isoproterenol, terbutaline, and dantrolene. Mean maximum twitch amplitude for 3 contractions/min was recorded and compared with predrug-vehicle values for the skeletal muscle segments, and area under the curve (AUC) for 3 contractions/min was compared with predrug-vehicle values for the smooth muscle segments. No drugs caused a significant change in skeletal muscle response. In smooth muscle, isoproterenol, terbutaline, and oxytocin significantly reduced AUC in a concentration-dependent manner. Maximum reduction in AUC was 69% at 10(-4) M for isoproterenol, 63% at 10(-6) M for terbutaline, and 64% at 10(-4) M for oxytocin. Isoproterenol, terbutaline, and oxytocin cause relaxation of the smooth muscle portion of the esophagus. The clinical relaxant effects on the proximal portion of the esophagus reported of drugs such as oxytocin, detomidine, and acepromazine may be the result of centrally mediated mechanisms.

Gastric Smooth Muscle Hamartomas Mimicking Polyps in a Dog: A Case Description and a Review of the Literature

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Marian A. Taulescu

2013-01-01

Full Text Available This report presents a case of two smooth muscle hamartomas of the stomach in a 10-year-old male Boxer. The clinical history of the animal was of chronic vomiting, weight loss, and intermittent gastric distension, and it died because of chronic and congestive heart failure. Gross, histology, and immunohistochemistry (IHC exams were performed. On necropsy, in the pyloric region of the stomach, two closely related polypoid growths between 10 and 15 mm in diameter were identified. On the cut sections, both polyps presented white to gray color, with homogenous architecture and well-defined limits. The thickness of the submucosal layer was seen to be increased to 1 cm. No other gastric alterations were identified by the necropsy exam. Histologically, both masses growth consisted of hyperplastic glands lined by foveolar epithelium, arranged in a papillary or branching pattern, and supported by a core of well-vascularised and marked smooth muscle tissue interspersed between glands. No dysplastic cells and mitotic figures were observed in these lesions. Immunohistochemistry revealed a strong cytoplasm labelling for smooth muscle actin of the bundles around the mucosal glands. To our knowledge, this is the first report of smooth muscle hamartomas mimicking multiple gastric polyps in dogs.

Effects of Gymnodinium breve toxin on the smooth muscle preparation of guinea-pig ileum

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Grunfeld, Y.; Spiegelstein, M.Y.

1974-01-01

1 The effects of Gymnodinium breve neurotoxin (GT) on smooth muscles were studied using the guinea-pig isolated ileum. 2 The toxin caused strong spasmogenic effects at 1-4 μg/ml, characterized by prolonged tonic contraction with superimposed pronounced pendular movements. Tachyphylaxis was observed upon administration of successive doses. 3 Atropine blocked the contractile response elicited by GT, whereas mepyramine and hexamethonium failed to do so. These findings tentatively suggested a cholinergic involvement at a post-ganglionic site of action. 4 In the presence of tetrodotoxin the effects of GT were abolished, excluding direct action of the toxin on the smooth muscle. 5 It is concluded that GT exerts its spasmogenic effects through stimulation of the post-ganglionic cholinergic nerve fibres. PMID:4155337

Smooth Muscle Tumor Originating in the Pleura: A Case Report and Updated Literature Review

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Santiago Fabián Moscoso Martínez

2016-01-01

Full Text Available Smooth muscle tumors (SMTs of the pleura are exceptionally rare. At present and to the best of these authors’ knowledge, there are only 17 cases reported in the literature. We describe a case of a 51-year-old woman who complained of left sided pleuritic chest pain. Further, computed tomography (CT revealed a left sided localized pleural-based mass involving the 9th rib. She underwent an interventional radiology guided percutaneous core biopsy of the lesion, which disclosed a “Smooth Muscle Tumor of Undetermined Malignant Potential (SMT-UMP.” A video-assisted thoracoscopic surgery (VATS was performed for diagnosis and treatment purposes. Resections of the pleural-based mass and 9th rib were performed. SMT-UMP was the definitive diagnosis.

Mixed endometrial stromal and smooth muscle tumor: report of a case with focal anaplasia and early postoperative lung metastasis.

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Shintaku, Masayuki; Hashimoto, Hiromi

2013-04-01

A rare case of a mixed endometrial stromal and smooth muscle tumor arising in the uterus of a 74-year-old woman is reported. The patient underwent hysterectomy for an enlarging uterine mass, and a large intramural tumor, showing marked central hyaline necrosis with calcification, was found. The tumor consisted of an admixture of a low-grade endometrial stromal sarcoma (ESS) and a fascicular proliferation of spindle cells suggesting smooth muscle differentiation, and a characteristic 'star-burst' appearance was found. In the ESS region, there were a few small foci of anaplasia where large polygonal cells with atypical nuclei and abundant eosinophilic cytoplasm proliferated, and the proliferative activity was locally increased in these foci. A small metastatic nodule appeared in the lung nine months after the hysterectomy, and the resected metastatic lesion showed features of anaplastic spindle cell sarcoma which was immunoreactive for CD10 but not for smooth muscle markers. Mixed endometrial stromal and smooth muscle tumors should be regarded as malignant neoplasms with the potential for hematogenous metastasis, particularly when they contain foci of cellular anaplasia. © 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

Anti-atherosclerotic plants which modulate the phenotype of vascular smooth muscle cells.

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Saleh Al-Shehabi, Tuqa; Iratni, Rabah; Eid, Ali H

2016-10-15

Cardiovascular disease (CVD) remains the leading cause of global death, with atherosclerosis being a major contributor to this mortality. Several mechanisms are implicated in the pathogenesis of this disease. A key element in the development and progression of atherosclerotic lesions is the phenotype of vascular smooth muscle cells. Under pathophysiologic conditions such as injury, these cells switch from a contractile to a synthetic phenotype that often possesses high proliferative and migratory capacities. Despite major advances made in the management and treatment of atherosclerosis, mortality associated with this disease remains high. This mandates that other approaches be sought. Herbal medicine, especially for the treatment of CVD, has been gaining more attention in recent years. This is in no small part due to the evidence-based values associated with the consumption of many plants as well as the relatively cheaper prices, easier access and conventional folk medicine "inherited" over generations. Sections: In this review, we provide a brief introduction about the pathogenesis of atherosclerosis then we highlight the role of vascular smooth muscle cells in this disease, especially when a phenotypic switch of these cells arises. We then thoroughly discuss the various plants that show potentially beneficial effects as anti-atherosclerotic, with prime attention given to herbs and plants that inhibit the phenotypic switch of vascular smooth muscle cells. Accumulating evidence provides the justification for the use of botanicals in the treatment or prevention of atherosclerosis. However, further studies, especially clinical ones, are warranted to better define several pharmacological parameters of these herbs, such as toxicity, tolerability, and efficacy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Experimental study on effect of arsenic trioxide on vascular smooth muscle cells

International Nuclear Information System (INIS)

Lu Qin; An Yanli; Niu Huanzhang; Teng Gaojun; Wang Zihao; Zhang Dongsheng; Fang Juanjuan

2007-01-01

Objective: To investigate the effect of arsenic trioxide (As 2 O 3 ) nanoparticles on rabbit vascular smooth muscle cells in vitro in comparison with normal form As 2 O 3 . Methods: The rabbit vascular smooth muscle cells were cultured in vitro. Nano and normal forms of As 2 O 3 with drug concentrations of 3 μmol/L were added into the cells. Cell proliferation curve was drawn according to the light absorption values of MTT test. Flow cytometry was applied to observe the apoptosis. DNA was extracted and underwent electrophoresis. Results: Cell proliferation treated with the 3 μmol/L concentration of As 2 O 3 was inhibited. Cell growth was inhibited markedly with increased treatment time, and the inhibition effect of nano drug form seemed stronger than that of normal form. MTT light absorption values of cells treated at 24, 48 and 72 h showed statistically significant difference (H=10.934, 15.039, 15.539, P 2 O 3 , normal drug form of As 2 O 3 and control group of cells without As 2 O 3 were 44.97%, 58.54%, 74.02% respectively. The early apoptosis rates were 16.89%, 11.27%, 11.20%, late apoptosis rates were 26.56%, 23.60%, 12.46%, and necrosis rates were 11.58%, 6.59%, 2.32% respectively. Agarose gel electrophoresis showed 'ladder' strand of DNA, with more strands and obscurity for nano drug form treated cells. Conclusion: Arsenic trioxide may inhibit the growth of rabbit vascular smooth muscle cells. The nano drug form showed stronger inhibition effect than that of the normal drug form. (authors)

Testosterone replacement maintains smooth muscle content in the corpus cavernosum of orchiectomized rats

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Graziele Halmenschlager

2017-10-01

Conclusion: Normal testosterone levels maintain CC smooth muscle content and do not influence elastic fibers, collagen content and apoptotic index. Further studies should be performed in order to investigate the mechanisms by which androgen mediates its effects on CC structure.

Phenotypic modulation of smooth muscle cells during formation of neointimal thickenings following vascular injury.

Science.gov (United States)

Thyberg, J

1998-07-01

Smooth muscle cells build up the media of mammalian arteries and constitute one of the principal cell types in atherosclerotic and restenotic lesions. Accordingly, they show a high degree of plasticity and are able to shift from a differentiated, contractile phenotype to a less differentiated, synthetic phenotype, and then back again. This modulation occurs as a response to vascular injury and includes a prominent structural reorganization with loss of myofilaments and formation of an extensive endoplasmic reticulum and a large Golgi complex. At the same time, the expression of cytoskeletal proteins and other gene products is altered. As a result, the cells lose their contractility and become able to migrate from the media to the intima, proliferate, and secrete extracellular matrix components, thereby contributing to the formation of intimal thickenings. The mechanisms behind this change in morphology and function of the smooth muscle cells are still incompletely understood. A crucial role has been ascribed to basement membrane proteins such as laminin and collagen type IV and adhesive proteins such as fibronectin. A significant role is also played by mitogenic proteins such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF). An improved knowledge of the regulation of smooth muscle differentiated properties represents an important part in the search for new methods of prevention and treatment of vascular disease.

Single Nisoldipine-Sensitive Calcium Channels in Smooth Muscle Cells Isolated from Rabbit Mesenteric Artery

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Worley, Jennings F.; Deitmer, Joachim W.; Nelson, Mark T.

1986-08-01

Single smooth muscle cells were enzymatically isolated from the rabbit mesenteric artery. At physiological levels of external Ca, these cells were relaxed and contracted on exposure to norepinephrine, caffeine, or high levels of potassium. The patch-clamp technique was used to measure unitary currents through single channels in the isolated cells. Single channels were selective for divalent cations and exhibited two conductance levels, 8 pS and 15 pS. Both types of channels were voltage-dependent, and channel activity occurred at potentials positive to -40 mV. The activity of both channel types was almost completely inhibited by 50 nM nisoldipine. These channels appear to be the pathways for voltage-dependent Ca influx in vascular smooth muscle and may be the targets of the clinically used dihydropyridines.

Role of chemical drive in recruiting upper airway and inspiratory intercostal muscles in patients with obstructive sleep apnea.

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Okabe, S; Chonan, T; Hida, W; Satoh, M; Kikuchi, Y; Takishima, T

1993-01-01

Upper airway dilating muscle activity increases during apneic episodes in patients with obstructive sleep apnea (OSA). To elucidate the relative contribution of chemical and nonchemical stimuli to augmentation of the upper airway dilating muscle, we measured the response of genioglossus muscle (GG) and inspiratory intercostal muscle (IIM) activities to obstructive apnea during non-REM sleep and compared them with the response to progressive hypoxia and hypercapnia during awake periods in seven male patients with OSA. GG EMG was measured with a wire electrode inserted percutaneously, and IIM EMG was measured with surface electrodes placed in the second intercostal space parasternally. Responses to hypoxia and to hypercapnia were assessed by rebreathing methods in the supine position while awake. Following these measurements, a sleep study was conducted with the EMG electrodes placed in the same locations. The relationship between GG and IIM activities during the cycle of apnea and postapneic ventilation in non-REM sleep was quasi-linear, and the slope of the regression line was significantly greater than those during progressive hypoxia and progressive hypercapnia. The amplitude of GG activity at 70% of maximum IIM activities in the hypoxic test was 140 +/- 20% (mean +/- SEM) during non-REM sleep, which was also significantly greater than that during hypoxia (51 +/- 10%) and that during hypercapnia (59 +/- 15%). These results suggest that nonchemical factors contribute considerably to augmentation of GG activity during obstructive apneic episodes. The nonchemical stimuli may arise from mechanoreceptors activated by upper airway obstruction and behavioral factors associated with change in sleep states.

Airway delivery of soluble factors from plastic-adherent bone marrow cells prevents murine asthma.

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Ionescu, Lavinia I; Alphonse, Rajesh S; Arizmendi, Narcy; Morgan, Beverly; Abel, Melanie; Eaton, Farah; Duszyk, Marek; Vliagoftis, Harissios; Aprahamian, Tamar R; Walsh, Kenneth; Thébaud, Bernard

2012-02-01

Asthma affects an estimated 300 million people worldwide and accounts for 1 of 250 deaths and 15 million disability-adjusted life years lost annually. Plastic-adherent bone marrow-derived cell (BMC) administration holds therapeutic promise in regenerative medicine. However, given the low cell engraftment in target organs, including the lung, cell replacement cannot solely account for the reported therapeutic benefits. This suggests that BMCs may act by secreting soluble factors. BMCs also possess antiinflammatory and immunomodulatory properties and may therefore be beneficial for asthma. Our objective was to investigate the therapeutic potential of BMC-secreted factors in murine asthma. In a model of acute and chronic asthma, intranasal instillation of BMC conditioned medium (CdM) prevented airway hyperresponsiveness (AHR) and inflammation. In the chronic asthma model, CdM prevented airway smooth muscle thickening and peribronchial inflammation while restoring blunted salbutamol-induced bronchodilation. CdM reduced lung levels of the T(H)2 inflammatory cytokines IL-4 and IL-13 and increased levels of IL-10. CdM up-regulated an IL-10-induced and IL-10-secreting subset of T regulatory lymphocytes and promoted IL-10 expression by lung macrophages. Adiponectin (APN), an antiinflammatory adipokine found in CdM, prevented AHR, airway smooth muscle thickening, and peribronchial inflammation, whereas the effect of CdM in which APN was neutralized or from APN knock-out mice was attenuated compared with wild-type CdM. Our study provides evidence that BMC-derived soluble factors prevent murine asthma and suggests APN as one of the protective factors. Further identification of BMC-derived factors may hold promise for novel approaches in the treatment of asthma.

The neuropeptide catestatin promotes vascular smooth muscle cell proliferation through the Ca{sup 2+}-calcineurin-NFAT signaling pathway

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Guo, Xiaoxia [Department of Cardiology, People' s Hospital, Peking University, No. 11 South Avenue, Xi Zhi Men Xicheng District, Beijing 100044 (China); Zhou, Chunyan, E-mail: chunyanzhou@bjmu.edu.cn [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191 (China); Sun, Ningling, E-mail: nlsun@263.net [Department of Cardiology, People' s Hospital, Peking University, No. 11 South Avenue, Xi Zhi Men Xicheng District, Beijing 100044 (China)

2011-04-22

Highlights: {yields} Catestatin stimulates proliferation of vascular smooth muscle cells in a dose-dependent manner. {yields} Catestatin provokes sustained increase in intracellular Ca{sup 2+}. {yields} Catestatin produces increased activation of calcineurin and promotes NFATc1 translocation into the nucleus. -- Abstract: The Chromogranin A-derived neuropeptide catestatin is an endogenous nicotinic cholinergic antagonist that acts as a pleiotropic hormone. Since catestatin shares several functions with other members derived from the chromogranin/secretogranin protein family and other neuropeptides which exert proliferative effects on vascular smooth muscle cells (VSMCs), we therefore hypothesized that catestatin would regulate VSMC proliferation. The present study demonstrates that catestatin caused a dose-dependent induction of proliferation in rat aortic smooth muscle cells and furthermore evoked a sustained increase in intracellular calcium. This subsequently leaded to enhanced activation of the Ca{sup 2+}/calmodulin-dependent phosphatase, calcineurin and resulted in an activation of the Ca{sup 2+}-dependent transcription factor, nuclear factor of activated T cells (NFAT), initiating transcription of proliferative genes. In addition, cyclosporin A (CsA), a potent inhibitor of calcineurin, abrogated catestatin-mediated effect on VSMCs, indicating that the calcineurin-NFAT signaling is strongly required for catestatin-induced growth of VSMCs. The present study establishes catestatin as a novel proliferative cytokine on vascular smooth muscle cells and this effect is mediated by the Ca{sup 2+}-calcineurin-NFAT signaling pathway.

Effect of aqueous extract of mango kernel (Mangifera Indica on basal activity of virgin rat uterine smooth muscle: role of muscarinic receptors

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Mehdi Noureddini

2017-04-01

Full Text Available Background: Mango belongs to the Anacardiaceae and the extracts from its stems, leaves, fruit and kernel are reported to affect smooth muscle contractility. We studied the role of cholinergic muscarinic receptors for the effects of aqueous extract of mango kernel (Mangifera indica on the basal activity of virgin rat uterine smooth muscle. Materials and Methods: In this experimental study, mid-sections (n=24 of the uterine of healthy virgin rats were placed in an organ bath containing carbonated Tyrode’s solution under 1 g tension. The cumulative effects of the aqueous extracts of mango kernel (0.002, 0.02, 0.2, 2, 20, 200, and 2000 μg/mL or extract vehicle (Tyrode’s solution in the presence or absence of atropine were examined by isometric method using the strength, frequency and contractile activity of uterine smooth muscle. Results: The cumulative concentrations (0.002-20 µg/ml of mango kernel aqueous extract was significantly decreased the strength, frequency and contractile activity of uterine smooth muscle, but the contractile activity was returned to the basal level at the concentrations of 200 and 2000 µg/ml. Atropine (1 µM could not significantly change the effects of cumulative use of extract on the strength and contractile activity of uterine smooth muscle, but it significantly enhanced the contractile frequency at low concentrations. Conclusion: The effects of aqueous extract of mango kernel on the activity of the uterine smooth muscle might not be through cholinergic muscarinic receptors and atropine could enhance the effects of the extract on frequency through other receptors.

Inhibition effect of Bifidobacterium longum, Lactobacillus acidophilus, Streptococcus thermophilus and Enterococcus faecalis and their related products on human colonic smooth muscle in vitro.

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Jing Gong

Full Text Available To investigate the effects of four strains, generally used in clinic, including Bifidobacterium longum, Lactobacillus acidophilus, Streptococcus thermophilus and Enterococcus faecalis, and their related products on human colonic smooth muscle in vitro.Human colonic circular muscle strips obtained from disease-free margins of resected segments from 25 patients with colorectal cancer were isometrically examined in a constant-temperature organ bath and exposed to different concentrations of living bacteria, sonicated cell fractions and cell-free supernatant (CFS. The area under the curve (AUC representing the contractility of smooth muscle strips was calculated.(1 The four living probiotics inhibited the contractility of human colonic muscle strips only at high concentration (1010 CFUs/mL, all P0.05.Four common probiotics related products, including the sonicated cell fractions and the CFS, obviously inhibited human colonic smooth muscles strips contraction in a dose-dependent manner. Only high concentration living probiotics (1010 CFUs/mL can inhibit the colonic smooth muscles strips contraction. The NO pathway may be partly involved in the inhibitory effect of CFS from Streptococcus thermophilus and Enterococcus faecalis.

[Mg2+, ATP-dependent plasma membrane calcium pump of smooth muscle cells. I. Structural organization and properties].

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Veklich, T O; Mazur, Iu Iu; Kosterin, S O

2015-01-01

Tight control of cytoplasm Ca2+ concentration is essential in cell functioning. Changing of Ca2+ concentration is thorough in smooth muscle cells, because it determines relaxation/constraint process. One of key proteins which control Ca2+ concentration in cytoplasm is Mg2+, ATP-dependent plasma membrane calcium pump. Thus, it is important to find compoumds which allowed one to change Mg2+, ATP-dependent plasma membrane calcium pump activity, as long as this topic is of current interest in biochemical research which regards energy and pharmacomechanical coupling mechanism of muscle excitation and contraction. In this article we generalized literatute and own data about properties of smooth muscle cell plasma membrane Ca(2+)-pump. Stuctural oganization, kinetical properties and molecular biology are considered.

Alteration of Airway Reactivity and Reduction of Ryanodine Receptor Expression by Cigarette Smoke in Mice.

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Donovan, Chantal; Seow, Huei Jiunn; Royce, Simon G; Bourke, Jane E; Vlahos, Ross

2015-10-01

Small airways are a major site of airflow limitation in chronic obstructive pulmonary disease (COPD). Despite the detrimental effects of long-term smoking in COPD, the effects of acute cigarette smoke (CS) exposure on small airway reactivity have not been fully elucidated. Balb/C mice were exposed to room air (sham) or CS for 4 days to cause airway inflammation. Changes in small airway lumen area in response to contractile agents were measured in lung slices in situ using phase-contrast microscopy. Separate slices were pharmacologically maintained at constant intracellular Ca(2+) using caffeine/ryanodine before contractile measurements. Gene and protein analysis of contractile signaling pathways were performed on separate lungs. Monophasic contraction to serotonin became biphasic after CS exposure, whereas contraction to methacholine was unaltered. This altered pattern of contraction was normalized by caffeine/ryanodine. Expression of contractile agonist-specific receptors was unaltered; however, all isoforms of the ryanodine receptor were down-regulated. This is the first study to show that acute CS exposure selectively alters small airway contraction to serotonin and down-regulates ryanodine receptors involved in maintaining Ca(2+) oscillations in airway smooth muscle. Understanding the contribution of ryanodine receptors to altered airway reactivity may inform the development of novel treatment strategies for COPD.

Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification.

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Moretti, Alessandra; Caron, Leslie; Nakano, Atsushi; Lam, Jason T; Bernshausen, Alexandra; Chen, Yinhong; Qyang, Yibing; Bu, Lei; Sasaki, Mika; Martin-Puig, Silvia; Sun, Yunfu; Evans, Sylvia M; Laugwitz, Karl-Ludwig; Chien, Kenneth R

2006-12-15

Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1(+) precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1(+) cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1(+)/Nkx2.5(+)/flk1(+) defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1(+) cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

Microscopic changes induced by Cr-VI in smooth muscles of albino mice

International Nuclear Information System (INIS)

Nabeel, H.

2007-01-01

Chromium is believed to be an essential trace element in human nutrition. Evidence suggests that it plays an important role in normal carbohydrate metabolism. It was found that patients receiving long-term total parenteral nutrition (TPN) without chromium developed glucose intolerance, weight loss and peripheral neuropathy Chromium is present in a normal diet at trace (but essential) levels. Occupational exposure is related to the industrial uses of chrome compounds in production and use of steels, pigments, leather tanning and wood preservation solutions, plating chemicals, and cement. Toxicity is predominantly associated with industrial exposures. Hexavalent chromium compounds appear to have greatest toxicity and almost all tissues of body are affected. To evaluate the effects on smooth muscles, present study was carried out. The mice of experimental group (2wks, 4wks, 6wks ,and 8wks) were injected Potassium dichromate (K/sub 2/Cr/sub 2/O/sub 7/) intraperitoneally according to experimental design. The drug caused slight to marked inflammation of smooth muscle fibers and vaculations of nuclei was also observed indicating degenerative changes. (author)

Mg(2+,ATP-dependent plasma membrane calcium pump of smooth muscle cells. ІІ. Regulation of activity

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T. О. Veklich

2015-04-01

Full Text Available Plasma membrane Ca2+-pump is one of key proteins, which takes part in Ca2+ exchange in smooth muscle cells. It has a lot of diverse functions from control of basal cytoplasmal Ca2+ concentration to regulation of proteins involved in Ca2+-dependent signal pathway. Ca2+ pump function is often depen­dent on the isoform or even form of alternative splicing. Allowing for a variety of Ca2+-pump functions and properties, which were reviewed in detail in the first part of our review article cycle (Ukr. Biochem. J., 2015; 87(1, the precise control of the mentioned pump activity is very important for cell functioning­. The other part of this article is dedicated to different regulation factors of smooth muscle plasma membrane Ca2+-pump activity: endogenous and exo­genous, biotic and abiotic factors. Special attention is given to literature data and own results about design and the search of selective plasma membrane Ca2+-pump inhibitor which would allow examining its functioning in smooth muscle cells more meticulously.

Smooth muscle cell phenotypic switching in stroke.

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Poittevin, Marine; Lozeron, Pierre; Hilal, Rose; Levy, Bernard I; Merkulova-Rainon, Tatiana; Kubis, Nathalie

2014-06-01

Disruption of cerebral blood flow after stroke induces cerebral tissue injury through multiple mechanisms that are not yet fully understood. Smooth muscle cells (SMCs) in blood vessel walls play a key role in cerebral blood flow control. Cerebral ischemia triggers these cells to switch to a phenotype that will be either detrimental or beneficial to brain repair. Moreover, SMC can be primarily affected genetically or by toxic metabolic molecules. After stroke, this pathological phenotype has an impact on the incidence, pattern, severity, and outcome of the cerebral ischemic disease. Although little research has been conducted on the pathological role and molecular mechanisms of SMC in cerebrovascular ischemic diseases, some therapeutic targets have already been identified and could be considered for further pharmacological development. We examine these different aspects in this review.

Recipient origin of neointimal vascular smooth muscle cells in cardiac allografts with transplant arteriosclerosis

NARCIS (Netherlands)

Hillebrands, JL; van den Hurk, BMH; Klatter, FA; Popa, ER; Nieuwenhuis, P; Rozing, J

2000-01-01

Background: Coronary artery disease is today's most important post-heart transplantation problem after the first perioperative year. Histologically, coronary artery disease is characterized by transplant arteriosclerosis. The current view on this vasculopathy is that vascular smooth muscle (VSM)

Actin isoform and alpha 1B-adrenoceptor gene expression in aortic and coronary smooth muscle is influenced by cyclical stretch.

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Lundberg, M S; Sadhu, D N; Grumman, V E; Chilian, W M; Ramos, K S

1995-09-01

The occurrence of vascular domains with specific biological and pharmacological characteristics suggests that smooth muscle cells in different arteries may respond differentially to a wide range of environmental stimuli. To determine if some of these vessel-specific differences may be attributable to mechano-sensitive gene regulation, the influence of cyclical stretch on the expression of actin isoform and alpha 1B-adrenoceptor genes was examined in aortic and coronary smooth muscle cells. Cells were seeded on an elastin substrate and subjected to maximal stretching (24% elongation) and relaxation cycles at a frequency of 120 cycles/min in a Flexercell strain unit for 72 h. Total RNA was extracted and hybridized to radiolabeled cDNA probes to assess gene expression. Stretch caused a greater reduction of actin isoform mRNA levels in aortic smooth muscle cells as compared to cells from the coronary artery. Steady-state mRNA levels of alpha 1B-adrenoceptor were also decreased by cyclical stretch in both cell types but the magnitude of the response was greater in coronary smooth muscle cells. No changes in alpha 1B-adrenoceptor or beta/gamma-actin steady-state mRNA levels were observed in H4IIE cells, a nonvascular, immortalized cell line. The relative gene expression of heat shock protein 70 was not influenced by the cyclic stretch regimen in any of these cell types. These results suggest that stretch may participate in the regulation of gene expression in vascular smooth muscle cells and that this response exhibits some degree of cell-specificity.

Retigabine diminishes the effects of acetylcholine, adrenaline and adrenergic agonists on the spontaneous activity of guinea pig smooth muscle strips in vitro.

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Apostolova, Elisaveta; Zagorchev, Plamen; Kokova, Vesela; Peychev, Lyudmil

2017-03-01

The aim of this study is to evaluate the effect of retigabine on the smooth muscle response to acetylcholine, adrenaline, α-and β-adrenoceptor agonists. We studied the change in the spontaneous smooth muscle contraction of guinea pig gastric corpus strips before and after 20-min treatment with 2μM retigabine. We also evaluated the effect of retigabine on the smooth muscle response to 10μM acetylcholine, 1 and 10μM adrenaline, 1μM methoxamine, 0.1μM p-iodoclonidine and 10μM isoproterenol. We observed a significant reduction in the effects of all studied mediators and agonists when they were added to organ baths in the presence of retigabine. Retigabine diminished the effect of acetylcholine on the spontaneous smooth muscle activity. The effect was fully antagonized by XE-991 (Kv7 channel blocker), which supports our hypothesis about the role of KCNQ channels in the registered changes. The increase in the contraction force after adding of 1μM adrenaline, methoxamine, and 0.1μM p-iodoclonidine was also significantly smaller in presence of retigabine. However, comparing the effect of 10μM adrenaline on the contractility before and after treatment with retigabine, we observed increased contractility when retigabine was present in the organ baths. A possible explanation for the observed diminished effects of mediators and receptor agonists is that the effect of retigabine on smooth muscle contractility is complex. The membrane hyperpolarization, the interaction between Kv7 channels and adrenoceptors, and the influence on signaling pathways may contribute to the summary smooth muscle response. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

International Nuclear Information System (INIS)

Petri, Marcelo H.; Tellier, Céline; Michiels, Carine; Ellertsen, Ingvill; Dogné, Jean-Michel; Bäck, Magnus

2013-01-01

Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A 2 is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Petri, Marcelo H. [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Tellier, Céline; Michiels, Carine [NARILIS, URBC, University of Namur, Namur (Belgium); Ellertsen, Ingvill [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Dogné, Jean-Michel [Department of Pharmacy, Namur Thrombosis and Hemostasis Center, University of Namur, Namur (Belgium); Bäck, Magnus, E-mail: Magnus.Back@ki.se [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)

2013-11-15

Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

Differentiated muscles are mandatory for gas-filling of the Drosophila airway system

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Yiwen Wang

2015-12-01

Full Text Available At the end of development, organs acquire functionality, thereby ensuring autonomy of an organism when it separates from its mother or a protective egg. In insects, respiratory competence starts when the tracheal system fills with gas just before hatching of the juvenile animal. Cellular and molecular mechanisms of this process are not fully understood. Analyses of the phenotype of Drosophila embryos with malformed muscles revealed that they fail to gas-fill their tracheal system. Indeed, we show that major regulators of muscle formation like Lame duck and Blown fuse are important, while factors involved in the development of subsets of muscles including cardiac and visceral muscles are dispensable for this process, suggesting that somatic muscles (or parts of them are essential to enable tracheal terminal differentiation. Based on our phenotypic data, we assume that somatic muscle defect severity correlates with the penetrance of the gas-filling phenotype. This argues that a limiting molecular or mechanical muscle-borne signal tunes tracheal differentiation. We think that in analogy to the function of smooth muscles in vertebrate lungs, a balance of physical forces between muscles and the elasticity of tracheal walls may be decisive for tracheal terminal differentiation in Drosophila.

Enhancement of S1P-induced contractile response in detrusor smooth muscle of rats having cystitis.

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Anjum, Irfan; Denizalti, Merve; Kandilci, Hilmi Burak; Durlu-Kandilci, Nezahat Tugba; Sahin-Erdemli, Inci

2017-11-05

Interstitial cystitis is a chronic disease characterized by lower abdominal pain and some nonspecific symptoms including an increase in urinary frequency and urgency. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that controls smooth muscle tone via G-protein coupled receptors (S1P 1-3 receptors). S1P production is known to take place both in physiological states and some pathological situations, such as in overactive bladder syndrome. The intracellular mechanism of S1P-induced contractile response was investigated in β-escin permeabilized detrusor smooth muscle of rats having cyclophosphamide-induced cystitis. The bladder was isolated from rats and detrusor smooth muscle strips were permeabilized with β-escin. S1P (50µM)-induced contraction and calcium sensitization response were significantly increased in cystitis. S1P-induced augmented contractile response was inhibited by S1P 2 receptor antagonist JTE-013 and S1P 3 receptor antagonist suramin. S1P 2 receptor protein expressions were increased in cystitis, where no change was observed in S1P 3 expressions between control and cystitis groups. S1P-induced contraction was reduced by Rho kinase (ROCK) inhibitor Y-27632 and protein kinase C (PKC) inhibitor GF-109203X in both control and cystitis group. S1P-induced increased calcium sensitization response was decreased by ROCK inhibitor and PKC inhibitor in cystitis. Our findings provide the first evidence that interstitial cystitis triggers S1P-induced increase in intracellular calcium in permeabilized detrusor smooth muscle of female rats. Both S1P 2 and S1P 3 receptors are involved in S1P mediated enhanced contractile response. The augmentation in S1P-induced contraction in interstitial cystitis involves both PKC and ROCK pathways of calcium sensitization. Copyright © 2017 Elsevier B.V. All rights reserved.

Nuclear import mechanism for myocardin family members and their correlation with vascular smooth muscle cell phenotype.

Science.gov (United States)

Nakamura, Seiji; Hayashi, Ken'ichiro; Iwasaki, Kazuhiro; Fujioka, Tomoaki; Egusa, Hiroshi; Yatani, Hirofumi; Sobue, Kenji

2010-11-26

Myocardin (Mycd), which is essential for the differentiation of the smooth muscle cell lineage, is constitutively located in the nucleus, although its family members, myocardin-related transcription factors A and B (MRTF-A/B), mostly reside in the cytoplasm and translocate to the nucleus in response to Rho signaling. The mechanism for their nuclear import is unclear. Here we investigated the mechanism for the nuclear import of Mycd family members and demonstrated any correlation between such mechanism and the phenotype of vascular smooth muscle cells (VSMCs). In cultured VSMCs, the knockdown of importin β1 inhibited the nuclear import of Mycd and MRTF-A/B. Their NH(2)-terminal basic domain was identified as a binding site for importin α/β1 by in vitro analyses. However, Mycd had a higher affinity for importin α/β1 than did MRTF-A/B, even in the absence of G-actin, and Mycd affinity for importin α1/β1 was stronger than for any other importin α/β1 heterodimers. The binding of Mycd to importin α/β1 was insensitive to G-actin, whereas that of MRTF-A/B was differently inhibited by G-actin. In dedifferentiated VSMCs, the levels of importins α1 and β1 were reduced concomitant with down-regulation of Mycd, serum response factor, and smooth muscle cell markers. By contrast, in differentiated VSMCs, their expressions were up-regulated. Thus, the nuclear import of Mycd family members in VSMCs depends on importin α/β1, and their relative affinities for importin α/β1 heterodimers determine Mycd nuclear import. The expression of Mycd nuclear import machineries is related to the expression levels of VSMC phenotype-dependent smooth muscle cell markers.

Eosinophils enhance WNT-5a and TGF-β1 genes expression in airway smooth muscle cells and promote their proliferation by increased extracellular matrix proteins production in asthma.

Science.gov (United States)

Januskevicius, Andrius; Vaitkiene, Simona; Gosens, Reinoud; Janulaityte, Ieva; Hoppenot, Deimante; Sakalauskas, Raimundas; Malakauskas, Kestutis

2016-06-13

Recent studies have suggested that eosinophils may have a direct effect on airway smooth muscle cells (ASMC), causing their proliferation in patients with asthma, but the precise mechanism of the interaction between these cells remains unknown. We propose that changes in Wnt signaling activity and extracellular matrix (ECM) production may help explain these findings. Therefore, the aim of this study was to investigate the effect of eosinophils from asthmatic and non-asthmatic subjects on Wnt-5a, transforming growth factor β1 (TGF-β1), and ECM protein (fibronectin and collagen) gene expression and ASMC proliferation. A total of 18 subjects were involved in the study: 8 steroid-free asthma patients and 10 healthy subjects. Peripheral blood eosinophils were isolated using centrifugation and magnetic separation. An individual co-culture of eosinophils with human ASMC was prepared for each study subject. Adhesion of eosinophils to ASMC (evaluated by assaying eosinophil peroxidase activity) was determined following various incubation periods (30, 45, 60, 120, and 240 min). The expression of Wnt-5a, TGF-β1, and ECM protein genes in ASMC was measured using quantitative real-time polymerase chain reaction (PCR) after 24 h of co-culture. Proliferation of ASMC was measured using the Alamar blue method after 48 h and 72 h of co-culture with eosinophils. Eosinophils from asthmatic subjects demonstrated increased adhesion to ASMC compared with eosinophils from healthy subjects (p eosinophils from asthmatic subjects, while co-culture of ASMC with eosinophils from healthy subjects increased only TGF-β1 and fibronectin gene expression. ASMC proliferation was augmented after co-culture with eosinophils from asthma patients compared with co-culture with eosinophils from healthy subjects (p Eosinophils enhance Wnt-5a, TGF-β1, fibronectin, and collagen gene expression in ASMC and promote proliferation of these cells in asthma. ClinicalTrials.gov Identifier: NCT02648074 .

Functional Modeling of the Shift in Cellular Calcium Dynamics at the Onset of Synchronization in Smooth Muscle Cells

DEFF Research Database (Denmark)

Postnov, D E; Brings Jacobsen, J C; von Holstein-Rathlou, Niels-Henrik

2011-01-01

In the present paper we address the nature of synchronization properties found in populations of mesenteric artery smooth muscle cells. We present a minimal model of the onset of synchronization in the individual smooth muscle cell that is manifested as a transition from calcium waves to whole......-cell calcium oscillations. We discuss how different types of ion currents may influence both amplitude and frequency in the regime of whole-cell oscillations. The model may also explain the occurrence of mixed-mode oscillations and chaotic oscillations frequently observed in the experimental system....

Intestinal smooth muscle response to chronic obstruction : possible applications in jejunoileal atresia.

Science.gov (United States)

Cloutier, R

1975-02-01

Hyperplasia is the main change occurring in intestinal smooth muscle above a chronic obstruction and explains the functional obstruction seen in the proximal bowel of a jejunoileal atresia. With an experimental model in dogs, this hyperplasia has been shown to be reversible. However, changes are extreme in atresia, and experiments in animals with induced atresia will best evaluate various kinds of treatment.

Human induced pluripotent stem cell-derived vascular smooth muscle cells

DEFF Research Database (Denmark)

Ayoubi, Sohrab; Sheikh, Søren P; Eskildsen, Tilde V

2017-01-01

. To this end, human induced pluripotent stem cells (hiPSCs) have generated great enthusiasm, and have been a driving force for development of novel strategies in drug discovery and regenerative cell-therapy for the last decade. Hence, investigating the mechanisms underlying the differentiation of hi......PSCs into specialized cell types such as cardiomyocytes, endothelial cells, and vascular smooth muscle cells (VSMCs) may lead to a better understanding of developmental cardiovascular processes and potentiate progress of safe autologous regenerative therapies in pathological conditions. In this review, we summarize...

Iduronic Acid in chondroitin/dermatan sulfate affects directional migration of aortic smooth muscle cells

NARCIS (Netherlands)

Bartolini, B.; Thelin, M.A.; Svensson, L.; Ghiselli, G.; Kuppevelt, T.H. van; Malmstrom, A.; Maccarana, M.

2013-01-01

Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS

Guiding the orientation of smooth muscle cells on random and aligned polyurethane/collagen nanofibers.

Science.gov (United States)

Jia, Lin; Prabhakaran, Molamma P; Qin, Xiaohong; Ramakrishna, Seeram

2014-09-01

Fabricating scaffolds that can simulate the architecture and functionality of native extracellular matrix is a huge challenge in vascular tissue engineering. Various kinds of materials are engineered via nano-technological approaches to meet the current challenges in vascular tissue regeneration. During this study, nanofibers from pure polyurethane and hybrid polyurethane/collagen in two different morphologies (random and aligned) and in three different ratios of polyurethane:collagen (75:25; 50:50; 25:75) are fabricated by electrospinning. The fiber diameters of the nanofibrous scaffolds are in the range of 174-453 nm and 145-419 for random and aligned fibers, respectively, where they closely mimic the nanoscale dimensions of native extracellular matrix. The aligned polyurethane/collagen nanofibers expressed anisotropic wettability with mechanical properties which is suitable for regeneration of the artery. After 12 days of human aortic smooth muscle cells culture on different scaffolds, the proliferation of smooth muscle cells on hybrid polyurethane/collagen (3:1) nanofibers was 173% and 212% higher than on pure polyurethane scaffolds for random and aligned scaffolds, respectively. The results of cell morphology and protein staining showed that the aligned polyurethane/collagen (3:1) scaffold promote smooth muscle cells alignment through contact guidance, while the random polyurethane/collagen (3:1) also guided cell orientation most probably due to the inherent biochemical composition. Our studies demonstrate the potential of aligned and random polyurethane/collagen (3:1) as promising substrates for vascular tissue regeneration. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Terbinafine: effects on platelet-derived growth factor-stimulated smooth muscle cells in vitro and myointimal proliferation in vivo

International Nuclear Information System (INIS)

McCarthy, L.; Van Halen, R.G.; St Denny, I.H.; Glinka, K.G.; Handley, D.A.; Stuetz, A.; Nemecek, G.M.

1987-01-01

Terbinafine (T; (E)-N-(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl-1-naphthalenemethanamine), an antimycotic agent with antimitogenic activity in fibroblasts, was examined for its effects on platelet-derived growth factor (PDGF)-stimulated aortic smooth muscle cell DNA synthesis in vitro and myointimal proliferation in vivo. Exposure of smooth muscle cells to 1-25 μM T resulted in a concentration-dependent inhibition of PDGF-induced mitogenesis as determined by [ 3 H]thymidine incorporation or cell number. The IC 50 for T was approximately 5 μM. The inhibitory effect of terbinafine persisted in the presence of 0.4-8.0 μg/ml cholesterol or 130 μg/ml mevalonate. Administration of T to rats for 2 d before and 14 d after balloon catheter carotid injury resulted in a 40% decrease in lesion area. These observations indicate that T is both a potent in vitro antagonist of the smooth muscle cell mitogenic response to PDGF and an effective, well-tolerated, orally active inhibitor of myointimal proliferation in vivo

Terbinafine: effects on platelet-derived growth factor-stimulated smooth muscle cells in vitro and myointimal proliferation in vivo

Energy Technology Data Exchange (ETDEWEB)

McCarthy, L.; Van Halen, R.G.; St. Denny, I.H.; Glinka, K.G.; Handley, D.A.; Stuetz, A.; Nemecek, G.M.

1987-05-01

Terbinafine (T; (E)-N-(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl-1-naphthalenemethanamine), an antimycotic agent with antimitogenic activity in fibroblasts, was examined for its effects on platelet-derived growth factor (PDGF)-stimulated aortic smooth muscle cell DNA synthesis in vitro and myointimal proliferation in vivo. Exposure of smooth muscle cells to 1-25 ..mu..M T resulted in a concentration-dependent inhibition of PDGF-induced mitogenesis as determined by (/sup 3/H)thymidine incorporation or cell number. The IC/sub 50/ for T was approximately 5 ..mu..M. The inhibitory effect of terbinafine persisted in the presence of 0.4-8.0 ..mu..g/ml cholesterol or 130 ..mu..g/ml mevalonate. Administration of T to rats for 2 d before and 14 d after balloon catheter carotid injury resulted in a 40% decrease in lesion area. These observations indicate that T is both a potent in vitro antagonist of the smooth muscle cell mitogenic response to PDGF and an effective, well-tolerated, orally active inhibitor of myointimal proliferation in vivo.

Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation

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Meurs Herman

2005-03-01

Full Text Available Abstract Background Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using guinea pig tracheal open-ring preparations, we now investigated the involvement of arginase in the modulation of neuronal nitric oxide synthase (nNOS-mediated relaxation induced by inhibitory nonadrenergic noncholinergic (iNANC nerve stimulation. Methods Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz-induced relaxation was measured in tracheal preparations precontracted to 30% with histamine, in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to the EFS-induced relaxation was assessed by the nonselective NOS inhibitor L-NNA (0.1 mM, while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor nor-NOHA (10 μM. Furthermore, the role of substrate availability to nNOS in EFS-induced relaxation was measured in the presence of various concentrations of exogenous L-arginine. Results EFS induced a frequency-dependent relaxation, ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz, which was inhibited with the NOS inhibitor L-NNA by 78.0 ± 10.5% at 0.5 Hz to 26.7 ± 7.7% at 8 Hz (P Conclusion The results indicate that endogenous arginase activity attenuates iNANC nerve-mediated airway relaxation by inhibition of NO generation, presumably by limiting L-arginine availability to nNOS.

Hypoxic contraction of cultured pulmonary vascular smooth muscle cells

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Murray, T.R.; Chen, L.; Marshall, B.E.; Macarak, E.J.

1990-01-01

The cellular events involved in generating the hypoxic pulmonary vasoconstriction response are not clearly understood, in part because of the multitude of factors that alter pulmonary vascular tone. The goal of the present studies was to determine if a cell culture preparation containing vascular smooth muscle (VSM) cells could be made to contract when exposed to a hypoxic atmosphere. Cultures containing only fetal bovine pulmonary artery VSM cells were assessed for contractile responses to hypoxic stimuli by two methods. In the first, tension forces generated by cells grown on a flexible growth surface (polymerized polydimethyl siloxane) were manifested as wrinkles and distortions of the surface under the cells. Wrinkling of the surface was noted to progressively increase with time as the culture medium bathing the cells was made hypoxic (PO2 approximately 25 mmHg). The changes were sometimes reversible upon return to normoxic conditions and appeared to be enhanced in cells already exhibiting evidence of some baseline tone. Repeated passage in culture did not diminish the hypoxic response. Evidence for contractile responses to hypoxia was also obtained from measurements of myosin light chain (MLC) phosphorylation. Conversion of MLC to the phosphorylated species is an early step in the activation of smooth muscle contraction. Lowering the PO2 in the culture medium to 59 mmHg caused a 45% increase in the proportion of MLC in the phosphorylated form as determined by two-dimensional gel electrophoresis. Similarly, cultures preincubated for 4 h with 32P and then exposed to normoxia or hypoxia for a 5-min experimental period showed more than twice as much of the label in MLCs of the hypoxic cells

Brain cytoplasmic RNA 1 suppresses smooth muscle differentiation and vascular development in mice.

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Wang, Yung-Chun; Chuang, Ya-Hui; Shao, Qiang; Chen, Jian-Fu; Chen, Shi-You

2018-04-13

The cardiovascular system develops during the early stages of embryogenesis, and differentiation of smooth muscle cells (SMCs) is essential for that process. SMC differentiation is critically regulated by transforming growth factor (TGF)-β/SMAD family member 3 (SMAD3) signaling, but other regulators may also play a role. For example, long noncoding RNAs (lncRNAs) regulate various cellular activities and events, such as proliferation, differentiation, and apoptosis. However, whether long noncoding RNAs also regulate SMC differentiation remains largely unknown. Here, using the murine cell line C3H10T1/2, we found that brain cytoplasmic RNA 1 (BC1) is an important regulator of SMC differentiation. BC1 overexpression suppressed, whereas BC1 knockdown promoted, TGF-β-induced SMC differentiation, as indicated by altered cell morphology and expression of multiple SMC markers, including smooth muscle α-actin (αSMA), calponin, and smooth muscle 22α (SM22α). BC1 appeared to block SMAD3 activity and inhibit SMC marker gene transcription. Mechanistically, BC1 bound to SMAD3 via RNA SMAD-binding elements (rSBEs) and thus impeded TGF-β-induced SMAD3 translocation to the nucleus. This prevented SMAD3 from binding to SBEs in SMC marker gene promoters, an essential event in SMC marker transcription. In vivo , BC1 overexpression in mouse embryos impaired vascular SMC differentiation, leading to structural defects in the artery wall, such as random breaks in the elastic lamina, abnormal collagen deposition on SM fibers, and disorganized extracellular matrix proteins in the media of the neonatal aorta. Our results suggest that BC1 is a suppressor of SMC differentiation during vascular development. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Resveratrol blocks interleukin-18-EMMPRIN cross-regulation and smooth muscle cell migration

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Venkatesan, Balachandar; Valente, Anthony J.; Reddy, Venkatapuram Seenu; Siwik, Deborah A.; Chandrasekar, Bysani

2009-01-01

Vascular smooth muscle cell (SMC) migration is an important mechanism in atherogenesis and postangioplasty arterial remodeling. Previously, we demonstrated that the proinflammatory cytokine interleukin (IL)-18 is a potent inducer of SMC migration. Since extracellular matrix metalloproteinase inducer (EMMPRIN) stimulates ECM degradation and facilitates cell migration, we investigated whether IL-18 and EMMPRIN regulate each other's expression, whether their cross talk induces SMC migration, and...

α-Trinositol inhibits FGF-stimulated growth of smooth muscle and breast cancer cells

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Siren, Matti J.; Vainiomaeki, Maija; Vaeaenaenen, Kalervo; Haerkoenen, Pirkko

2004-01-01

α-Trinositol (D-myo-inositol-1,2,6-trisphosphate), an isomer of the intracellular messenger IP 3 , has been studied for its anti-inflammatory and other effects in animal experiments and in human. The mechanisms of action remain unknown. Several human pathologies are associated with uncontrolled production of fibroblast growth factors (FGFs). FGF-2 induces vascular smooth muscle cell proliferation, which contributes to restenosis after coronary balloon angioplasty. The expression of several FGFs is also increased in tumors. We studied the effects of the water- and lipid-soluble derivatives of α-trinositol on the FGF-2- and/or FGF-8-induced proliferation of human pulmonary artery smooth muscle cells (HPASMC) and S115 mouse breast cancer cells. α-Trinositol decreased the FGF-mediated proliferation of HPASMC and S115 cells. Membrane permeability did not seem obligatory since the lipid-soluble form of α-trinositol was less effective than the water-soluble derivative. These results suggest a new biological function for certain phosphoinositides in the modulation of FGF-regulated processes

IGF-1 Has Plaque-Stabilizing Effects in Atherosclerosis by Altering Vascular Smooth Muscle Cell Phenotype

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von der Thüsen, Jan H.; Borensztajn, Keren S.; Moimas, Silvia; van Heiningen, Sandra; Teeling, Peter; van Berkel, Theo J.C.; Biessen, Erik A.L.

2011-01-01

Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic plaque on IGF-1 signaling and stability-related phenotypic parameters of murine vSMCs in vitro, and the effects of IGF-1 supplementation on plaque phenotype in an atherosclerotic mouse model. M1-polarized, macrophage-conditioned medium inhibited IGF-1 signaling by ablating IGF-1 and increasing IGF-binding protein 3, increased vSMC apoptosis, and decreased proliferation. Expression of α-actin and col3a1 genes was strongly attenuated by macrophage-conditioned medium, whereas expression of matrix-degrading enzymes was increased. Importantly, all of these effects could be corrected by supplementation with IGF-1. In vivo, treatment with the stable IGF-1 analog Long R3 IGF-1 in apolipoprotein E knockout mice reduced stenosis and core size, and doubled cap/core ratio in early atherosclerosis. In advanced plaques, Long R3 IGF-1 increased the vSMC content of the plaque by more than twofold and significantly reduced the rate of intraplaque hemorrhage. We believe that IGF-1 in atherosclerotic plaques may have a role in preventing plaque instability, not only by modulating smooth muscle cell turnover, but also by altering smooth muscle cell phenotype. PMID:21281823

Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels

DEFF Research Database (Denmark)

Chen, Xiaoping; Yang, Dachun; Ma, Shuangtao

2010-01-01

Vasomotion describes oscillations of arterial vascular tone due to synchronized changes of intracellular calcium concentrations. Since increased calcium influx into vascular smooth muscle cells from spontaneously hypertensive rats (SHR) has been associated with variances of transient receptor pot...

Intratracheal Administration of Mesenchymal Stem Cells Modulates Tachykinin System, Suppresses Airway Remodeling and Reduces Airway Hyperresponsiveness in an Animal Model.

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Konrad Urbanek

Full Text Available The need for new options for chronic lung diseases promotes the research on stem cells for lung repair. Bone marrow-derived mesenchymal stem cells (MSCs can modulate lung inflammation, but the data on cellular processes involved in early airway remodeling and the potential involvement of neuropeptides are scarce.To elucidate the mechanisms by which local administration of MSCs interferes with pathophysiological features of airway hyperresponsiveness in an animal model.GFP-tagged mouse MSCs were intratracheally delivered in the ovalbumin mouse model with subsequent functional tests, the analysis of cytokine levels, neuropeptide expression and histological evaluation of MSCs fate and airway pathology. Additionally, MSCs were exposed to pro-inflammatory factors in vitro.Functional improvement was observed after MSC administration. Although MSCs did not adopt lung cell phenotypes, cell therapy positively affected airway remodeling reducing the hyperplastic phase of the gain in bronchial smooth muscle mass, decreasing the proliferation of epithelium in which mucus metaplasia was also lowered. Decrease of interleukin-4, interleukin-5, interleukin-13 and increase of interleukin-10 in bronchoalveolar lavage was also observed. Exposed to pro-inflammatory cytokines, MSCs upregulated indoleamine 2,3-dioxygenase. Moreover, asthma-related in vivo upregulation of pro-inflammatory neurokinin 1 and neurokinin 2 receptors was counteracted by MSCs that also determined a partial restoration of VIP, a neuropeptide with anti-inflammatory properties.Intratracheally administered MSCs positively modulate airway remodeling, reduce inflammation and improve function, demonstrating their ability to promote tissue homeostasis in the course of experimental allergic asthma. Because of a limited tissue retention, the functional impact of MSCs may be attributed to their immunomodulatory response combined with the interference of neuropeptide system activation and tissue

Ayurvedic preparation of Zingiber officinale Roscoe: effects on cardiac and on smooth muscle parameters.

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Leoni, Alberto; Budriesi, Roberta; Poli, Ferruccio; Lianza, Mariacaterina; Graziadio, Alessandra; Venturini, Alice; Broccoli, Massimiliano; Micucci, Matteo

2017-08-28

The rhizome of the Zingiber officinale Roscoe, a biennial herb growing in South Asia, is commonly known as ginger. Ginger is used in clinical disorders, such as constipation, dyspepsia, diarrhoea, nausea and vomiting and its use is also recommended by the traditional medicine for cardiopathy, high blood pressure, palpitations and as a vasodilator to improve the circulation. The decoction of ginger rhizome is widely used in Ayurvedic medicine. In this papery by high-performance liquid chromatography, we have seen that its main phytomarkers were 6-gingerol, 8-gingerol and 6-shogaol and we report the effects of the decoction of ginger rhizome on cardiovascular parameters and on vascular and intestinal smooth muscle. In our experimental models, the decoction of ginger shows weak negative inotropic and chronotropic intrinsic activities but a significant intrinsic activity on smooth muscle with a potency on ileum is greater than on aorta: EC 50  = 0.66 mg/mL versus EC 50  = 1.45 mg/mL.

Role of pp60(c-src) and p(44/42) MAPK in ANG II-induced contraction of rat tonic gastrointestinal smooth muscles.

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Puri, Rajinder N; Fan, Ya-Ping; Rattan, Satish

2002-08-01

We examined the role of mitogen-activated protein kinase (p(44/42) MAPK) in ANG II-induced contraction of lower esophageal sphincter (LES) and internal anal sphincter (IAS) smooth muscles. Studies were performed in the isolated smooth muscles and cells (SMC). ANG II-induced changes in the levels of phosphorylation of different signal transduction and effector proteins were determined before and after selective inhibitors. ANG II-induced contraction of the rat LES and IAS SMC was inhibited by genistein, PD-98059 [a specific inhibitor of MAPK kinases (MEK 1/2)], herbimycin A (a pp60(c-src) inhibitor), and antibodies to pp60(c-src) and p(120) ras GTPase-activating protein (p(120) rasGAP). ANG II-induced contraction of the tonic smooth muscles was accompanied by an increase in tyrosine phosphorylation of p(120) rasGAP. These were attenuated by genistein but not by PD-98059. ANG II-induced increase in phosphorylations of p(44/42) MAPKs and caldesmon was attenuated by both genistein and PD-98059. We conclude that pp60(c-src) and p(44/42) MAPKs play an important role in ANG II-induced contraction of LES and IAS smooth muscles.

LC/MS/MS data analysis of the human uterine smooth muscle S-nitrosoproteome fingerprint in pregnancy, labor, and preterm labor

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Craig C. Ulrich

2015-09-01

Full Text Available The data described in this article is the subject of an article in the American Journal of Physiology: Cell Physiology, titled “The Human Uterine Smooth Muscle S-nitrosoproteome Fingerprint in Pregnancy, Labor, and Preterm Labor” (doi:10.1152/ajpcell.00198.2013 (Ulrich et al., 2013 [1]. The data described is a large scale mass spectrometry data set that defines the human uterine smooth muscle S-nitrosoproteome differences among laboring, non-laboring, preterm laboring tissue after treatment with S-nitrosoglutathione.

Smooth muscle enfoldment internal sphincter construction after intersphincteric resection for rectal cancer.

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Heiying Jin

Full Text Available To assess smooth muscle enfoldment and internal sphincter construction (SMESC for improvement of continence after intersphincteric resection (ISR for rectal cancer.Twenty-four Bama miniature pigs were randomly divided into a conventional ISR group and experimental SMESC group, with 12 pigs in each group. The proximal sigmoid colon was anastomosed directly to the anus in the ISR group. In the SMESC group, internal sphincter construction was performed. At 12 weeks before and after surgery, rectal resting pressure and anal canal length were assessed. Three-dimensional ultrasound was used to determine the thickness of the internal sphincter. After the animals were sacrificed, the rectum and anus were resected and pathological examinations were performed to evaluate the differences in sphincter thickness and muscle fibers.All 24 animals in the SMESC group and the ISR group survived the surgery. Twelve weeks post-surgery, the rectal resting pressure, length of the anal high-pressure zone and the postoperative internal sphincter thickness for the ISR group were significantly lower than for the SMESC group. There was a thickened area (about 2 cm above the anastomotic stoma among animals from the SMESC group; in addition, the smooth muscles were significantly enlarged and enfolded when compared to the ISR group.This animal model study shows that the SMESC procedure achieved acceptable reconstruction of the internal anal neo-sphincter (IAN/S, without increasing surgical risk. However, the findings in this experimental animal model must be confirmed by clinical trials to determine the safety and efficacy of this procedure in clinical practice.

Smooth muscle enfoldment internal sphincter construction after intersphincteric resection for rectal cancer.

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Jin, Heiying; Zhang, Bei; Yao, Hang; Du, Yonghong; Wang, Xiaofeng; Leng, Qiang

2014-01-01

To assess smooth muscle enfoldment and internal sphincter construction (SMESC) for improvement of continence after intersphincteric resection (ISR) for rectal cancer. Twenty-four Bama miniature pigs were randomly divided into a conventional ISR group and experimental SMESC group, with 12 pigs in each group. The proximal sigmoid colon was anastomosed directly to the anus in the ISR group. In the SMESC group, internal sphincter construction was performed. At 12 weeks before and after surgery, rectal resting pressure and anal canal length were assessed. Three-dimensional ultrasound was used to determine the thickness of the internal sphincter. After the animals were sacrificed, the rectum and anus were resected and pathological examinations were performed to evaluate the differences in sphincter thickness and muscle fibers. All 24 animals in the SMESC group and the ISR group survived the surgery. Twelve weeks post-surgery, the rectal resting pressure, length of the anal high-pressure zone and the postoperative internal sphincter thickness for the ISR group were significantly lower than for the SMESC group. There was a thickened area (about 2 cm) above the anastomotic stoma among animals from the SMESC group; in addition, the smooth muscles were significantly enlarged and enfolded when compared to the ISR group. This animal model study shows that the SMESC procedure achieved acceptable reconstruction of the internal anal neo-sphincter (IAN/S), without increasing surgical risk. However, the findings in this experimental animal model must be confirmed by clinical trials to determine the safety and efficacy of this procedure in clinical practice.

The structure of the actin-smooth muscle myosin motor domain complex in the rigor state.

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Banerjee, Chaity; Hu, Zhongjun; Huang, Zhong; Warrington, J Anthony; Taylor, Dianne W; Trybus, Kathleen M; Lowey, Susan; Taylor, Kenneth A

2017-12-01

Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I, -II and -V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops. Here we report a ∼6 Å resolution reconstruction of F-actin decorated with the nucleotide-free recombinant smooth muscle myosin-II motor domain (MD) from images recorded using a direct electron detector. Resolution is highest for F-actin and the actin-myosin interface (3.5-4 Å) and lowest (∼6-7 Å) for those parts of the MD at the highest radius. Atomic models built into the F-actin density are quite comparable to those previously reported for rabbit muscle actin and show density from the bound ADP. The atomic model of the MD, is quite similar to a recently published structure of vertebrate non-muscle myosin II bound to F-actin and a crystal structure of nucleotide free myosin-V. Larger differences are observed when compared to the cryoEM structure of F-actin decorated with rabbit skeletal muscle myosin subfragment 1. The differences suggest less closure of the 50 kDa domain in the actin bound skeletal muscle myosin structure. Copyright © 2017 Elsevier Inc. All rights reserved.

Lipopolysaccharide does not alter small airway reactivity in mouse lung slices.

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Donovan, Chantal; Royce, Simon G; Vlahos, Ross; Bourke, Jane E

2015-01-01

The bacterial endotoxin, lipopolysaccharide (LPS) has been associated with occupational airway diseases with asthma-like symptoms and in acute exacerbations of COPD. The direct and indirect effects of LPS on small airway reactivity have not been fully elucidated. We tested the hypothesis that both in vitro and in vivo LPS treatment would increase contraction and impair relaxation of mouse small airways. Lung slices were prepared from naïve Balb/C mice and cultured in the absence or presence of LPS (10 μg/ml) for up to 48 h for measurement of TNFα levels in conditioned media. Alternatively, mice were challenged with PBS or LPS in vivo once a day for 4 days for preparation of lung slices or for harvest of lungs for Q-PCR analysis of gene expression of pro-inflammatory cytokines and receptors involved in airway contraction. Reactivity of small airways to contractile agonists, methacholine and serotonin, and bronchodilator agents, salbutamol, isoprenaline and rosiglitazone, were assessed using phase-contrast microscopy. In vitro LPS treatment of slices increased TNFα release 6-fold but did not alter contraction or relaxation to any agonists tested. In vivo LPS treatment increased lung gene expression of TNFα, IL-1β and ryanodine receptor isoform 2 more than 5-fold. However there were no changes in reactivity in lung slices from these mice, even when also incubated with LPS ex vivo. Despite evidence of LPS-induced inflammation, neither airway hyperresponsiveness or impaired dilator reactivity were evident. The increase in ryanodine receptor isoform 2, known to regulate calcium signaling in vascular smooth muscle, warrants investigation. Since LPS failed to elicit changes in small airway reactivity in mouse lung slices following in vitro or in vivo treatment, alternative approaches are required to define the potential contribution of this endotoxin to altered small airway reactivity in human lung diseases.

Lipopolysaccharide does not alter small airway reactivity in mouse lung slices.

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Chantal Donovan

Full Text Available The bacterial endotoxin, lipopolysaccharide (LPS has been associated with occupational airway diseases with asthma-like symptoms and in acute exacerbations of COPD. The direct and indirect effects of LPS on small airway reactivity have not been fully elucidated. We tested the hypothesis that both in vitro and in vivo LPS treatment would increase contraction and impair relaxation of mouse small airways. Lung slices were prepared from naïve Balb/C mice and cultured in the absence or presence of LPS (10 μg/ml for up to 48 h for measurement of TNFα levels in conditioned media. Alternatively, mice were challenged with PBS or LPS in vivo once a day for 4 days for preparation of lung slices or for harvest of lungs for Q-PCR analysis of gene expression of pro-inflammatory cytokines and receptors involved in airway contraction. Reactivity of small airways to contractile agonists, methacholine and serotonin, and bronchodilator agents, salbutamol, isoprenaline and rosiglitazone, were assessed using phase-contrast microscopy. In vitro LPS treatment of slices increased TNFα release 6-fold but did not alter contraction or relaxation to any agonists tested. In vivo LPS treatment increased lung gene expression of TNFα, IL-1β and ryanodine receptor isoform 2 more than 5-fold. However there were no changes in reactivity in lung slices from these mice, even when also incubated with LPS ex vivo. Despite evidence of LPS-induced inflammation, neither airway hyperresponsiveness or impaired dilator reactivity were evident. The increase in ryanodine receptor isoform 2, known to regulate calcium signaling in vascular smooth muscle, warrants investigation. Since LPS failed to elicit changes in small airway reactivity in mouse lung slices following in vitro or in vivo treatment, alternative approaches are required to define the potential contribution of this endotoxin to altered small airway reactivity in human lung diseases.

Notch signaling regulates platelet-derived growth factor receptor-β expression in vascular smooth muscle cells

NARCIS (Netherlands)

Jin, S.; Hansson, E.M.; Tikka, S.; Lanner, F.; Sahlgren, C.; Farnebo, F.; Baumann, M.; Kalimo, H.; Lendahl, U.

2008-01-01

Notch signaling is critically important for proper architecture of the vascular system, and mutations in NOTCH3 are associated with CADASIL, a stroke and dementia syndrome with vascular smooth muscle cell (VSMC) dysfunction. In this report, we link Notch signaling to platelet-derived growth factor

Dipeptidyl peptidase-4 inhibitor gemigliptin protects against vascular calcification in an experimental chronic kidney disease and vascular smooth muscle cells.

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Soon-Youn Choi

Full Text Available Although dipeptidyl peptidase-4 inhibitors, a class of antidiabetic drugs, have various pleiotropic effects, it remains undetermined whether gemigliptin has a beneficial effect on vascular calcification. Therefore, this study was performed to evaluate the effect of gemigliptin on vascular calcification in a rat model of adenine-induced chronic kidney disease and in cultured vascular smooth muscle cells. Gemigliptin attenuated calcification of abdominal aorta and expression of RUNX2 in adenine-induced chronic kidney disease rats. In cultured vascular smooth muscle cells, phosphate-induced increase in calcium content was reduced by gemigliptin. Gemigliptin reduced phosphate-induced PiT-1 mRNA expression, reactive oxygen species generation, and NADPH oxidase mRNA expression (p22phox and NOX4. The reduction of oxidative stress by gemigliptin was associated with the downregulation of phospho-PI3K/AKT expression. High phosphate increased the expression of frizzled-3 (FDZ3 and decreased the expression of dickkopf-related protein-1 (DKK-1 in the Wnt pathway. These changes were attenuated by gemigliptin treatment. Gemigliptin restored the decreased expression of vascular smooth muscle cells markers (α-SMA and SM22α and increased expression of osteogenic makers (CBFA1, OSX, E11, and SOST induced by phosphate. In conclusion, gemigliptin attenuated vascular calcification and osteogenic trans-differentiation in vascular smooth muscle cells via multiple steps including downregulation of PiT-1 expression and suppression of reactive oxygen species generation, phospho-PI3K/AKT, and the Wnt signaling pathway.

IGF-1 has plaque-stabilizing effects in atherosclerosis by altering vascular smooth muscle cell phenotype

NARCIS (Netherlands)

von der Thüsen, Jan H.; Borensztajn, Keren S.; Moimas, Silvia; van Heiningen, Sandra; Teeling, Peter; van Berkel, Theo J. C.; Biessen, Erik A. L.

2011-01-01

Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic

CD16(+) monocytes with smooth muscle cell characteristics are reduced in human renal chronic transplant dysfunction

NARCIS (Netherlands)

Boersema, M.; van den Born, Joost; van Ark, J.; Harms, Geertruida; Seelen, M. A.; van Dijk, M. C. R. F.; van Goor, H.; Navis, G. J.; Popa, E. R.; Hillebrands, J. L.

In chronic transplant dysfunction (CTD), persistent (allo)immune-mediated inflammation eventually leads to tissue remodeling including neointima formation in intragraft arteries. We previously showed that recipient-derived neointimal alpha-SMA(+) smooth muscle-like cells are present in human renal

Influence of 103Pd radioactive stent on apoptosis of vascular smooth muscle cells

International Nuclear Information System (INIS)

Liu Yingmei; Wu Wei; Chen Xiaochao; Zhang Xuming; Wang Jingfeng; Wei Yulin; Yang Li

2003-01-01

Objective: To evaluate the influence of 103 Pd radioactive stent on apoptosis and its relative genes bcl-2 and bax in injured vascular media smooth muscle cells of rabbit abdominal arteries and to investigate the mechanism of 103 Pd radioactive stent for preventing restenosis after angioplasty. Methods: Fifty male New Zealand rabbits were randomized into stent group and 103 Pd stent group. Each group was subdivided into 5 sub-groups. Control group was set up. The study arteries were harvested at 3, 7, 14, 28 and 56 d after stenting and the pathomorphology, apoptosis analysis and in situ hybridization were performed to evaluate the expression of bcl-2 and bax mRNA. Results: The severity of the restenosis in 103 Pd stent group was less than that of stent group. It was most obvious at the 56th day (P 103 Pd stent group had much more apoptosis of vascular smooth muscle cells than stent group did and reached the peak at the 7th day, (14.72±0.53)% vs (12.42±1.13)% (P 103 Pd stent group was much lower than that of stent group at 3 to 28 d. The difference was most obvious at the 28th day after stenting, (18.43± 0.67)% vs (21.55±0.93)% (P 103 Pd stent group was higher than that of stent group, the peak was at the 7th day, (11.17±0.94)% vs (9.30±1.01)%. The ratio of bcl-2/bax in 103 Pd stent group was much lower than that of stent group at 3 to 28 d. Linear correlation analysis showed that there was significant negative correlation between bcl-2 mRNA and apoptosis. Between bax mRNA and apoptosis, the positive correlation was found (P 103 Pd radioactive stent induced more significant apoptosis in vascular media smooth muscle cells by promoting the expression of apoptosis related genes and relieved the expanding of restenosis

Tetraspanin CD9 regulates cell contraction and actin arrangement via RhoA in human vascular smooth muscle cells.

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